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rhegmatogenous retinal detachment (rrd) is characterized by the accumulation of subretinal fluid between the neurosensory retina and retinal pigment epithelium following the formation of a retinal break. the pathogenesis of rrd is complex and incompletely understood, involving age - related and/or inherited structural and molecular changes of the vitreous extracellular matrix and vitreoretinal interface, and the process of posterior vitreous detachment. the annual incidence of the condition has been estimated at 12.05 per 100,000, and although primary surgical reattachment is successful in the great majority of cases, photoreceptor cell death, subretinal fibrosis, and proliferative vitreoretinopathy (pvr) continue to be significant causes of reduced visual outcomes. pvr involves the proliferation and migration of various cell types including retinal pigment epithelial (rpe) cells, mller cells, inflammatory cells, and hyalocytes, which contribute to the formation of vitreal and periretinal membranes that can impede photoreceptor regeneration following surgical reattachment and cause tractional retinal detachment. the postulated epithelial - to - mesenchymal transition of rpe cells and mller cell activation and growth onto the retinal surfaces are believed to be pivotal events in pvr. it appears that the exposure of such cells to the vitreous and associated growth factors as a of rrd significantly contributes to the pathogenesis of pvr, though the basic cause as well as a clinically effective therapeutic approach for this condition remains elusive. proteomics studies proteins on a large scale in pursuit of a global and integrated view of disease processes at the protein level, which may potentially lead to the identification of novel biomarkers and therapeutic targets useful in clinical practice. rrd would likely be associated with alterations in the proteomic profiles of both the retina and vitreous. indeed, we initially undertook the first such retinal study from which a number of potentially important proteins were identified. the present study extends the proteomic investigation to the vitreous of this rabbit model of retinal detachment, building upon previous such analyses of human vitreous , in order to add further knowledge of the underlying pathophysiology. inferior retinal detachment was created in the right eyes of six new zealand red pigmented rabbits. combined injections of xylazine (6.7 mg / kg) and ketamine (33.3 mg / kg) were administered intramuscularly to induce anesthesia and analgesia. the pupils were dilated with topical drops of atropine and tropicamide (1% solutions). a pipette tip, with an external diameter of approximately 100 m, sodium hyaluronate (healon, 0.25% in a balanced salt solution ; pharmacia, piscataway, nj) was infused via a glass pipette between the neurosensory retina and retinal pigment epithelium. healon was necessary to prevent spontaneous retinal reattachment, and 0.25% is the most dilute solution that maintains the detachment for extended periods. approximately 50% of the retina beneath the medullary rays, which included the central retina, was detached (figure 1). sham surgery was performed in the right eyes of five other rabbits that were used as controls, which involved surgical entry of the vitreous cavity without disruption of the retina. seven days postoperatively the animals were euthanized by the administration of sodium pentobarbital (120 mg / kg ; butler schein, dublin, oh) and the eyes enucleated. after removal of the cornea and lens, the associated vitreous of the sham and detached retinas was extracted and immediately snap - frozen in liquid nitrogen within separate vials. there was no gross evidence of blood or other contamination of the vitreous samples at the time of tissue harvesting. all of the animal experiments undertaken in this study were in accordance with the standards of the national institutes of health animal care and use committee protocols, the arvo statement for the use of animals in ophthalmic and vision research, and the guidelines of the animal resource center, university of california, santa barbara. the rabbit vitreous samples were homogenized and dissolved in a lysis buffer containing 9 m urea, 2% (v / v) triton x-100, 2% (v / v) immobilized ph gradient (ipg) buffer (ph 310 nonlinear), and 2% (w / v) dithiothreitol (dtt). the total protein content in each vitreous sample was determined with non - interfering protein assay (calbiochem, san diego, ca). the extracted proteins were first fractionated by isoelectric focusing (ief) using ph 310 nonlinear 18 cm ipg strips (ge healthcare, chalfont st . the ipg strips were rehydrated for 20 h at room temperature in 200 l lysis buffer each containing 20 g protein from individual vitreous samples and 150 l rehydration buffer ( 8 m urea, 2% ( w / v) 3--1-propanesulfonate (chaps), 0.3% (w / v) dtt, and 2% (v / v) ipg buffer ), using the immobiline drystrip reswelling tray (ge healthcare). the ief was undertaken on a multiphor ii electrophoresis system (ge healthcare) at 500 v for 5 h and 3500 v in two steps for 5 h and 9.5 h in a gradient mode at 17c with the use of a multitemp iii thermostatic circulator (ge healthcare). before the second - dimension sodium dodecyl sulfate (sds) polyacrylamide gel electrophoresis (page), the ipg strips were equilibrated firstly for 10 min with gentle agitation in 20 ml of equilibration solution (0.6% ( w / v) tris - hcl, ph 6.8, 6 m urea, 30% (v / v) glycerol, 1% (w / v) sds, and 0.05% (w / v) dtt ) and secondly using 4.5% (w / v) iodoacetamide and bromophenol blue. the ipg strips were then transferred to 12% polyacrylamide gels for electrophoresis, which was performed at a maximum voltage of 50 v for approximately 20 h to separate the proteins vertically on the basis of molecular mass. the two - dimensional (2d) gels were silver stained using a protocol optimized for protein identification with mass spectrometry. in brief, the gels were fixed overnight in 50% (v / v) ethanol, 12% (v / v) acetic acid, and 0.0185% (v / v) formaldehyde. the gels were washed 3 times for 20 min in 35% (v / v) ethanol and pretreated for 1 min in 0.02% (w / v) na2s2o35h2o. they were then rinsed in water and stained for 20 min in 0.2% (w / v) agno3 and 0.028% (v / v) formaldehyde. following further rinsing with water, development was undertaken for approximately 3 min in 6% (w / v) na2co3, 0.0185% (v / v) formaldehyde, and 0.0004% (w / v) na2s2o35h2o. the development was arrested in a fixative solution of 40% (v / v) ethanol and 12% (v / v) acetic acid. silver stained 2d gels were scanned on a gs-710 calibrated imaging densitometer (bio - rad, hercules, ca) using the quantity one program (bio - rad), and the pdquest software (bio - rad) was used to define, quantify, and match the protein spots on each of the 2d gels. all well - defined protein spots that were at least twofold (mann - whitney u test, p < 0.05) differentially expressed between the sham and retinal detachment vitreous groups were selected for identification with nanoliquid chromatography - electrospray ionization tandem mass spectrometry (lc - ms / ms). the selected protein spots were carefully excised from the gels with a scalpel and subjected to in - gel digestion with trypsin gold (mass spectrometry grade ; promega, madison, wi). the peptide samples that were obtained were analyzed by lc - ms / ms as previously described. in brief, peptides generated by trypsin digestion were separated on an inert nano - lc system (lc packings, san francisco, ca) connected to a q - tof premier mass spectrometer (waters, milford, ma). the masslynx 4 sp4 (waters) was used to obtain spectra and the raw data was processed using proteinlynx global server 2.1 (waters). the processed data were used to search the total part of the swiss - prot database using the online version of the mascot ms / ms ions search facility (matrix science, ltd .). the search was undertaken with doubly and triply charged ions with up to two missed cleavages, a peptide tolerance of 50 ppm, one variable modification, carbamidomethyl - c, and a ms / ms tolerance of 0.05 da. contaminating peptides such as trypsin, keratin, bovine serum albumin, and all peptides originating from previous samples were disregarded. individual peptide ions scores above approximately 36 indicated identity or extensive homology giving a less than 5% probability that the observed match was a random event. all peptides for the protein hits are reported (table 1). in each case three micrograms of vitreous sample protein was separated on novex 1020% gradient tris - glycine polyacrylamide gels (invitrogen corporation, carlsbad, ca) and subsequently transferred to nitrocellulose hybond - c extra membranes (ge healthcare). the membranes were blocked overnight with 5% skimmed milk in 80 mm na2hpo4, 20 mm nah2po4, 100 mm nacl, and 0.05% tween 20 buffer, ph 7.5. membranes were incubated with anti - albumin (genway biotech, ca, usa ; 1 : 5000) and anti - peroxiredoxin 2 (abcam, cambridge, uk ; 1 : 200). no suitable antibodies were commercially available for the rabbit f isoform of -1-antiproteinase or the rabbit collagen - i1 fragment that was identified with lc - ms / ms. following washing , the membranes were further incubated with appropriate horseradish peroxidase - conjugated secondary antibodies: p0163 sheep and p0260 mouse (both 1 : 1000 ; dako, glostrup, denmark). proteins were visualized with the enhanced chemiluminescence system (ge healthcare) and imaging system (fujifilm las-3000, tokyo, japan). up to approximately 340 protein spots were clearly resolved on each of the 11 2d gels. ten protein spots were found to be significantly and at least twofold differentially expressed between the sham and detachment vitreous groups (figure 2). three protein spots were upregulated and seven spots were downregulated. from the three upregulated protein spots, two were identified as fragments of albumin (spots 5104 and 6101), whilst spot 6205 could not be identified. four of the seven downregulated protein spots were identified as fragment of collagen - i1 (spot 0503), -1-antiproteinase f (spots 0703 and 1707), and peroxiredoxin 2 (spot 0705). protein spots 0102, 0815, and 1302 could not be identified (figure 2 ; table 1). western blotting developed with anti - albumin showed a heavy band at approximately 60 kda, which is likely to represent the full length protein, whilst multiple bands below this suggest the presence of several fragments, some of which may correspond with those identified with the 2d - page analysis (figure 3, left). however, spot 0705 containing peroxiredoxin 2 migrates with a molecular mass around 60 kda with 2d - page (figure 2), and this size was verified by western blot analysis (figure 3, right). though a single and specific band was achieved with anti - peroxiredoxin 2, western blotting could not be reliably used for quantification due to a weak signal near the detection limit and variable reaction. analysis with 2d - page revealed fragments of albumin to be upregulated in the vitreous following retinal detachment. albumin is the most abundant protein in plasma, aqueous, and vitreous humor, where in the latter it constitutes around 6070% of total protein. serum proteins such as albumin are present in the aqueous and vitreous humor at a relatively lower level compared to the vascular circulation from where they may have in part originated. western blot analysis showed an intense band at approximately 60 kda corresponding with the full length albumin protein, with multiple lower molecular mass bands that are likely its fragments. increase of albumin and its fragments may signify increased proteolysis and the passage of albumin into the vitreous. indeed, the breakdown of the blood - retinal barrier that occurs with retinal detachment has also been implicated in the increase of other such proteins in the vitreous. it is also possible that albumin in the vitreous may arise from de novo synthesis in the retina, similar to the reported increased gene and protein expression of albumin in the corneal epithelium during wound healing. extraocular albumin is known to have diverse and important functions, which include maintenance of colloid osmotic pressure, transport of biomolecules, and inactivation of toxins through intermolecular binding. albumin can also act as an antioxidant by scavenging reactive oxygen species and sequestration of metal ions and has anti - inflammatory and apoptotic regulatory abilities. vitreal albumin has been proposed to transport long chain fatty acids into the lens for biosynthesis of lenticular lipids. indeed, albumin is likely to have many such important roles in the eye, which requires further investigation. in the present study we observed peroxiredoxin 2 to have a molecular mass above 60 kda using both 2d - page and western blot analyses. however, the predicted molecular mass of the peroxiredoxin family of proteins is approximately 22 kda31 kda. this variation may represent the well - studied property of these proteins to undergo oligomerization, which can be promoted by a number of factors including overoxidation of cysteine residues of peroxiredoxin. although the present experiments were conducted in standard reducing conditions that aim to break cysteine bonds, we obtained a band well above 20 kda. this is in keeping with another study, which also showed some peroxiredoxin 2 western blot bands appearing at molecular mass much higher than 20 kda that was suggested to from oligomerization or posttranslational modification. our finding could also represent a novel alternative splicing variant of peroxiredoxin 2, as reported for peroxiredoxin 5. the peroxiredoxins are a group of ubiquitous antioxidant proteins that currently comprise six members in mammals. these proteins are primarily found at high levels intracellularly, mainly within the cytosol, but are also present in the mitochondria, peroxisomes, and nuclei, and they may be exported. furthermore, presence of peroxiredoxin 2 has been shown in plasma, not only as a of hemolysis but also possibly by secretion from the t lymphocytes. these multifunction enzymes act as antioxidants by using redox active cysteines for the reduction and degradation of hydrogen peroxide, peroxynitrite, and organic hydroperoxides. oxidative stress is thought to from an imbalance between reactive oxygen species production and antioxidant ability and is recognized to be an important factor in the pathogenesis of a number of age - related and neurodegenerative diseases, which include age - related cataract, age - related macular degeneration, glaucoma, diabetic retinopathy, retinal detachment, and pvr. indeed, the present study showed a decrease in the vitreal levels of peroxiredoxin 2 following retinal detachment. this may be in keeping with reported reductions in the levels of other members of the antioxidant defense system such as glutathione and ascorbic acid both in vitreal and in blood samples of patients suffering from pvr. furthermore, apart from their role as antioxidants, the peroxiredoxins can affect a diverse range of biological processes that include cellular proliferation, differentiation, and apoptosis by influencing signal transduction pathways that employ hydrogen peroxide as a secondary messenger. recent studies on tears from patients with glaucoma have also identified peroxiredoxin 1 as having a possible involvement in inflammation. indeed, peroxiredoxin 2 and other members of this family of proteins are liable to have a significant role in the pathophysiology of retinal detachment. a fragment of collagen - i1 was identified in the vitreous of the rabbit; however, type i collagen has not previously been identified as a natural component of the mammalian vitreous and is rather known to be a constituent of early pvr membranes and retinal blood vessels. a mixture of type ii, ix, and v / xi hybrid collagen fibrils, which are separated out mainly by water and ions attracted to hyaluronan, characterizes the vitreous body. collagen, possibly with the aid of adhesive - like intermediate molecules, may provide the basis of vitreoretinal adhesion by connecting the vitreous with the retinal inner limiting membrane (ilm). this attachment is extremely strong in the vitreous base since the fibrils pass through the ilm to merge into underlying collagen networks and crypts. collagen is also a significant component of both epiretinal and subretinal pvr membranes , and type i collagen is recognized to be a principal constituent during their early development. the presence of collagen in the subretinal space, a place normally devoid of this protein, suggests that certain cells, particularly the rpe and mller cells associated with membranes, are able to synthesize collagen under certain pathological conditions such as retinal detachment and pvr. however, the present analysis suggests collagen - i1 fragment to be found in sham vitreous, which furthermore showed a decreased concentration following retinal detachment that may indicate perturbed proteolytic activity. matrix metalloproteinases (mmp) and other proteolytic enzymes that are able to degrade and remodel vitreal collagen have been found to be increased with rrd and pvr , which could be in keeping with the decrease in -1-antiproteinase shown in the present study. further studies will be necessary to confirm the source and nature of collagen - i1 in the vitreous and the possible mechanisms of collagen fragmentation that may be an important feature of vitreous liquefaction and rrd. 2d - page showed -1-antiproteinase (also called -1-antitrypsin or -1-proteinase inhibitor) at two closely positioned spots, which were largely in keeping with their predicted molecular mass but differing by their charge. currently, four isoforms of -1-antiproteinase have been identified in the rabbit, termed f, s1, s2, and e, which is a similar picture to the multiple variants identified in humans. alpha-1-antiproteinase is an acute phase protein and archetypal member of the superfamily of serine protease inhibitors (serpin), which are involved in a wide range of biological processes that includes inflammation, angiogenesis, blood coagulation, ecm remodeling, and tumor suppression. this protein has the ability to inhibit a large number of serine proteases though its principle target is neutrophil elastase. indeed, -1-antiproteinase originally received much attention because its deficiency increases the risk of a variety of clinical conditions, such as chronic obstructive pulmonary disease, which can from unrestrained elastase activity. we found the f isoform of rabbit -1-antiproteinase to be downregulated in the vitreous following retinal detachment. so far identified that has been shown to have the oxidizable methionine residue site that is present in human -1-antiproteinase. the oxidation of methionine to methionine sulfoxide, which can occur during episodes of inflammation as a of oxygen - free radicals secreted by leucocytes, has an inhibiting effect upon -1-antiproteinase function. this process is thought to enhance the ability of proteinases such as elastase to locally degrade tissue debris that occurs at sites of inflammation. alpha-1-antiproteinase is primarily produced in the liver and circulated to the rest of the body tissues via the blood; however, extrahepatic sites of its synthesis have been identified, which include blood monocytes, alveolar macrophages, bronchial and gastrointestinal epithelial cells, and the cornea. the protein has also been localized to the tear film, aqueous humor, and vitreous, where in the latter a phosphorylated form of -1-antiproteinase has been suggested as a potential biomarker of idiopathic macular hole and rhegmatogenous retinal detachment. it has been postulated that one of the main functions of corneal -1-antiproteinase is to protect against the damaging effects of neutrophil elastase produced during corneal inflammation, and it may be expected that a similar role in addition to others is applicable to vitreal -1-antiproteinase, though this requires further investigation. this proteomic investigation of the rabbit vitreous has identified a set of proteins that assist our understanding of the pathogenesis of rhegmatogenous retinal detachment and its complications. certain proteins, such as those of low abundance and at the extremes of molecular mass, together with membrane proteins, can be difficult to resolve and detect using the 2d - page technique. therefore, complementary proteomic methods such as gel - free mass spectrometry should be considered in future work in order to help address these limitations.

purpose. the pathogenesis of rhegmatogenous retinal detachment (rrd) remains incompletely understood, with no clinically effective treatment for potentially severe complications such as photoreceptor cell death and proliferative vitreoretinopathy. here we investigate the protein profile of the vitreous following experimental retinal detachment using a comparative proteomic based approach. materials and methods. retinal detachment was created in the right eyes of six new zealand red pigmented rabbits. sham surgery was undertaken in five other rabbits that were used as controls. after seven days the eyes were enucleated and the vitreous was removed. the vitreous samples were evaluated with two - dimensional polyacrylamide gel electrophoresis and the differentially expressed proteins were identified with tandem mass spectrometry. . ten protein spots were found to be at least twofold differentially expressed when comparing the vitreous samples of the sham and retinal detachment surgery groups. protein spots that were upregulated in the vitreous following retinal detachment were identified as albumin fragments, and those downregulated were found to be peroxiredoxin 2, collagen - i1 fragment, and -1-antiproteinase f. . proteomic investigation of the rabbit vitreous has identified a set of proteins that help further our understanding of the pathogenesis of rhegmatogenous retinal detachment and its complications.

primary breast lymphoma (pbl) is a rare neoplasm that accounts for 0.4% of malignant breast lesions and 2% of extranodal lymphomas. diagnosis of pbl by fine - needle aspiration cytology (fnac) is reported infrequently. a 23-year - old non - lactating (para 2, gravida 0) lady came with a left breast lump and lymphedema of the left arm of 1-month duration. she recently had lumpectomy done in the same breast 1-month earlier at a peripheral hospital, which was diagnosed as breast abscess on histopathology. her ultrasound examination previous to lumpectomy was available that showed an irregular hypoechoic mass of size 2.6 2.1 1.6 cm in the left breast having both solid and cystic components, perilesional inflammatory changes, and architectural distortion. the right breast was normal and bilateral axillary lymphadenopathy was absent at this point of time. her examination at the present visit revealed an irregular hard tender fixed mass of 6 4 cm in the outer lower and upper quadrants of the left breast. the skin overlying the mass was erythematous, nodular, and showed a surgical scar mark just above the areola. fnac of the left breast mass and left axillary lymph nodes was done in department of pathology. the breast mass fnac showed richly cellular monomorphic population of loosely cohesive single lymphoid cells of two to three times the size of mature lymphocytes with moderate anisonucleosis, 1 - 3 prominent nucleoli and fragile cytoplasm. similar cytomorphologic features were also present in the aspirate from left axillary lymph nodes. a cytological diagnosis of non - hodgkin lymphoma- large cell type was given. a tru - cut needle biopsy of the breast mass also showed features of non - hodgkin lymphoma. her formalin - fixed, paraffin - embedded blocks of the prior lumpectomy specimen was then retrieved from the peripheral laboratory and reviewed. hematoxylin and eosin (h and e)-stained sections showed replacement of the breast parenchyma by diffuse sheets of monomorphic lymphoid cells. on higher magnification, the lymphoid cells showed moderate anisokaryosis, clumped nuclear chromatin, prominent nucleoli, and minimal amount of eosinophilic cytoplasm on a of lymphoglandular bodies. lymphoepithelial lesion was characterized by infiltration of lymphoma cells into the sparsely present duct epithelium of the breast. immunohistochemical studies were performed on formalin fixed, paraffin - embedded blocks of the lumpectomy specimen. cytokeratin immunostaining showed strong cytoplasmic reactivity in the duct epithelium while the lymphoma cells stained negative. in contrast, the lymphoma cells expressed diffuse strong membranous positivity with antibodies against leukocyte common antigen and cd 20 while negative for cd 15, cd 30, and cd 3. there was no primary elsewhere as evidenced by the absence of hepatosplenomegaly and the absence of any lymph node enlargement other than left axillary lymph nodes. the final diagnosis was primary diffuse large b - cell lymphoma of the breast, stage ii. the patient was planned for chemotherapy, her baseline laboratory investigations and ultrasound examination of abdomen was normal. however, further management and follow up of the patient could not be done as she escaped from the hospital against the medical advice. (b) fnac showing dispersed large non - cleaved lymphoma cells aspirated from the breast lump (pap, x400). (c) diffuse infiltration of the lymphoma cells ing in destruction and fibrosis of breast parenchyma (h and e, x200). (d) magnified view of lymphoma cells against a of lymphoglandular bodies (h and e, x400) (a) lymphoepithelial lesion demonstrating invasion of ductal epithelium of the breast by lymphoma cells (h and e, x200). (b) cytokeratin immunostaining strongly positive in ductal epithelium, negative in surrounding lymphoma cells (ihc, x200). (c) diffuse strong lca positive immunostaining in tumour cells (ihc, x400). (d) lymphoma cells showing diffuse strong positivity against cd 20 (ihc, x400) the deceptive benign appearance on clinical and ultrasound examinations was diagnosed as non - hodgkin lymphoma. fnac provided the earliest clue to diagnosis of pbl which was later confirmed by histology and immunohistochemistry in this case. because breast lymphoma is a rare condition, however, clinician should be aware of the deceiving benign appearance of pbl in contrast to much more commonly seen breast carcinoma. on physical examination, pbl often appears as a rapidly growing single palpable nodule of mean size 3.5 cm that is mobile and non - tender. this probably from the relatively rapid growth of these lesions in comparison to breast carcinomas, especially in the younger patients.. clinical signs of advanced breast malignancy are rare, such as inflammatory changes, nipple retraction, puckering of the skin or tumor fixation. breast lymphoma can occasionally present as a diffuse rapid breast enlargement in the younger age group, or as breast skin thickening due to lymphatic blockage by lymphoma ing in retrograde edema. on diagnostic mammography , pbl often appears as a well - defined mass with smooth margins and homogeneous appearance. again, signs of breast malignancy such as microcalcification, spiculation, and distortion of surrounding tissue are usually absent. likewise pbl will most often demonstrate a well - defined hypoechoic lesion without enhancement suggesting a wrong notion of benignity. the specific criteria for diagnosis of pbl were first defined by wiseman and liao in 1972. these are: (a) the clinical site of appearance is the breast; (b) a history of prior lymphoma or evidence of widespread disease are absent at diagnosis; (c) demonstration of lymphoma in proximity to breast tissue; and (d) involvement of ipsilateral lymph nodes may be present if they develop simultaneously with the primary breast tumor. the entire criteria for diagnosis of pbl are satisfactorily present in our case. at the time of initial lumpectomy, however, when patient came to us she had significant enlargement of the ipsilateral axillary lymph nodes in association with the recurring breast lump (stage ii disease). this offers evidence that excision biopsy alone is not curative in patients with pbl; rather such a procedure may upstage the disease process in these patients. thus, if a young patient presents with a rapidly growing breast tumor, lymphoma should be considered before undertaking any surgical intervention. in such cases, a meticulously performed fnac is vital for an accurate preoperative diagnosis of the breast lymphoma. in summary , pbl is a rare entity that accounts for only a small fraction of the breast malignancies. as none of the clinical or imaging features of the disease is characteristic, a rapidly growing breast lump in a young female should warn the attending clinician of this rare possibility. fnac is very useful, simple, rapid, reliable, and cost - effective procedure for an accurate diagnosis of the lesion.

primary breast lymphoma (pbl) is a rare entity. it represents 0.4% of malignant breast lesions and 2% of extranodal lymphomas. a 23-year - old woman presented with a left breast lump followed by palpable left axillary lymphadenopathy. fine - needle aspiration cytology (fnac) and histopathology were diagnostic of non - hodgkin lymphoma - large cell type. immunohistochemistry was positive against leukocyte common antigen and cd20. because pbls are uncommon malignant lesions and they usually do not have characteristic clinical and imaging findings, fnac may prove to be a simple, rapid, reliable, and cost - effective procedure for successful diagnosis of the pbl.

the study was approved by the research ethics boards of the university of ottawa and ottawa hospital. two of the participants were competitive athletes training 6 days per week, while those remaining were recreationally active. all participants had been regularly performing both aerobic and resistance exercise at least three times weekly for a minimum of 6 months. participants were using either multiple daily injections (mdis) of insulin or continuous subcutaneous insulin infusion with an insulin pump. the same cohort of participants also took part in a previously published study from the same research group. testing took place in the human and environmental physiology research unit at the university of ottawa. participants provided written informed consent prior to being tested for vo2max, muscular strength (eight repetition maximum), and hba1c as previously described. the cgms system gold (medtronic, northridge, ca) was used in this study so that participants would be blinded to their glucose values and would not change their behavior based on real - time glucose monitoring. onetouch ultrasmart handheld glucose meters (lifescan ; johnson & johnson, milpitas, ca) and coded strips (same code throughout the study) were provided for capillary glucose tests. twenty - four hours after the end of the exercise / no - exercise control session, cgm units were retrieved and data were downloaded (minimed solutions v.3.0c ; medtronic, northridge, ca). over each monitoring period, participants consumed the same self - selected breakfast, lunch, and dinner daily at the same times of day and recorded food and insulin intake on study log sheets. participants refrained from exercise for 24 h before insertion of the sensor (48 h before the experimental session) and avoided caffeine and alcohol during the monitoring period. participants arrived at the laboratory at 4:00 p.m. on the day after the sensor insertion. the following sessions were performed, separated by at least 5 days: 1 ) resistance exercise, three sets of eight repetitions maximum of seven different exercises with 90-s rest between sets (duration 45 min); 2 ) aerobic exercise, 45 min of treadmill exercise (60% of vo2max); and 3 ) no - exercise control, 45 min of seated rest. testing sessions for the female participants, who were using monophasic oral contraceptives, took place during the active pill - consumption phase. participants reduced their insulin doses on exercise days by making either a 10% decrease in intermediate or long - acting insulin (mdi) or a 50% decrease in basal rate starting 1 h before exercise and maintained until the end of exercise for pump users. if blood glucose was < 5 mmol / l upon arrival, those using insulin pumps decreased their basal rate a further 25%. participants consumed a standard snack (glucerna chocolate graham snack bars, 150 calories, 25 g carbohydrate ; abbott laboratories, abbott park, il) at 4:00 p.m. every day, including the exercise day, with the bar consumed upon arrival at the laboratory. capillary glucose was checked 60 and 30 min before exercise and immediately prior to exercise to ensure glucose levels 5.5 and 13.9 mmol / l. venous blood samples were collected through an intravenous catheter at baseline and 5, 10, 15, 30, and 45 min during all three testing sessions (resistance exercise, aerobic exercise, and no - exercise control) and at the 50-, 55-, 60-, 65-, 75-, 85-, 95-, and 105-min blood was immediately mixed by inversion, centrifuged (4,000 revolutions / min for 4 min), and stored at 80c. the hexokinase timed end point method was used to determine plasma glucose levels using the beckman coulter unicel dxc600 synchron clinical analyzer (beckman coulter, fullerton, ca) and synchron cx systems glucose reagent (cat . glucose levels were compared among sessions using two - way repeated - measures ( time and condition) anova. exercise and recovery periods were examined separately among the three sessions (aerobic, resistance, and no - exercise control). the exercise period consisted of the 5-, 10-, 15-, 30-, and 45-min time points, while the recovery period consisted of the remaining time points. paired sample t tests were used to perform pairwise post hoc comparisons for each time point between conditions (aerobic, resistance, or no - exercise control) within exercise and recovery separately and to examine changes from baseline and changes from the end of exercise within each exercise condition. cgm data were examined as 15-min averages in the following windows: 24-h pre - exercise, overnight (12:00 a.m. to 6:00 a.m.) pre - exercise, 16 h postexercise, overnight postexercise, and 24 h postexercise. a two - way (time and condition) repeated - measures anova was used to compare among conditions in the 16-h postexercise period. paired sample t tests were then used to perform pairwise post hoc comparisons for each 15-min segment. the minimum, maximum, and mean blood glucose; amount of time spent in hypoglycemic and hyperglycemic states; and areas under the curve (aucs) for time spent in hypo- and hyperglycemic states were determined for each window. pre - exercise values were compared with postexercise values within exercise conditions using related - samples wilcoxon signed rank tests. differences among conditions were examined using related - samples friedman two - way anova by ranks. agreement between cgm data and capillary glucose over the 3 days was determined by performing pearson correlations between sensor glucose and self - recorded capillary glucose values. daily total insulin and carbohydrate intake was calculated based on the information provided in participant logs. comparisons among conditions for each day were made using related - samples friedman two - way anova by ranks. where significant were found, related - samples wilcoxon signed rank tests ensued for determination of where the differences lie. analyses were performed using spss 18.0 for windows (spss, chicago, il). testing took place in the human and environmental physiology research unit at the university of ottawa. participants provided written informed consent prior to being tested for vo2max, muscular strength (eight repetition maximum), and hba1c as previously described. the cgms system gold (medtronic, northridge, ca) was used in this study so that participants would be blinded to their glucose values and would not change their behavior based on real - time glucose monitoring. onetouch ultrasmart handheld glucose meters (lifescan ; johnson & johnson, milpitas, ca) and coded strips (same code throughout the study) were provided for capillary glucose tests. twenty - four hours after the end of the exercise / no - exercise control session, cgm units were retrieved and data were downloaded (minimed solutions v.3.0c ; medtronic, northridge, ca). over each monitoring period , participants consumed the same self - selected breakfast, lunch, and dinner daily at the same times of day and recorded food and insulin intake on study log sheets. participants refrained from exercise for 24 h before insertion of the sensor (48 h before the experimental session) and avoided caffeine and alcohol during the monitoring period. participants arrived at the laboratory at 4:00 p.m. on the day after the sensor insertion. the following sessions were performed, separated by at least 5 days: 1 ) resistance exercise, three sets of eight repetitions maximum of seven different exercises with 90-s rest between sets (duration 45 min); 2 ) aerobic exercise, 45 min of treadmill exercise (60% of vo2max); and 3 ) no - exercise control, 45 min of seated rest. testing sessions for the female participants, who were using monophasic oral contraceptives, took place during the active pill - consumption phase. participants reduced their insulin doses on exercise days by making either a 10% decrease in intermediate or long - acting insulin (mdi) or a 50% decrease in basal rate starting 1 h before exercise and maintained until the end of exercise for pump users. if blood glucose was < 5 mmol / l upon arrival, those using insulin pumps decreased their basal rate a further 25%. participants consumed a standard snack (glucerna chocolate graham snack bars, 150 calories, 25 g carbohydrate ; abbott laboratories, abbott park, il) at 4:00 p.m. every day, including the exercise day, with the bar consumed upon arrival at the laboratory. capillary glucose was checked 60 and 30 min before exercise and immediately prior to exercise to ensure glucose levels 5.5 and 13.9 mmol / l. venous blood samples were collected through an intravenous catheter at baseline and 5, 10, 15, 30, and 45 min during all three testing sessions (resistance exercise, aerobic exercise, and no - exercise control) and at the 50-, 55-, 60-, 65-, 75-, 85-, 95-, and 105-min time points during recovery. blood was immediately mixed by inversion, centrifuged (4,000 revolutions / min for 4 min), and stored at 80c. the hexokinase timed end point method was used to determine plasma glucose levels using the beckman coulter unicel dxc600 synchron clinical analyzer (beckman coulter, fullerton, ca) and synchron cx systems glucose reagent (cat . glucose levels were compared among sessions using two - way repeated - measures ( time and condition) anova. exercise and recovery periods were examined separately among the three sessions (aerobic, resistance, and no - exercise control). the exercise period consisted of the 5-, 10-, 15-, 30-, and 45-min time points, while the recovery period consisted of the remaining time points. paired sample t tests were used to perform pairwise post hoc comparisons for each time point between conditions (aerobic, resistance, or no - exercise control) within exercise and recovery separately and to examine changes from baseline and changes from the end of exercise within each exercise condition. cgm data were examined as 15-min averages in the following windows: 24-h pre - exercise, overnight (12:00 a.m. to 6:00 a.m.) pre - exercise, 16 h postexercise, overnight postexercise, and 24 h postexercise. a two - way (time and condition) repeated - measures anova was used to compare among conditions in the 16-h postexercise period. paired sample t tests were then used to perform pairwise post hoc comparisons for each 15-min segment. the minimum, maximum, and mean blood glucose; amount of time spent in hypoglycemic and hyperglycemic states; and areas under the curve (aucs) for time spent in hypo- and hyperglycemic states were determined for each window. pre - exercise values were compared with postexercise values within exercise conditions using related - samples wilcoxon signed rank tests. differences among conditions were examined using related - samples friedman two - way anova by ranks. agreement between cgm data and capillary glucose over the 3 days was determined by performing pearson correlations between sensor glucose and self - recorded capillary glucose values. daily total insulin and carbohydrate intake was calculated based on the information provided in participant logs. comparisons among conditions for each day were made using related - samples friedman two - way anova by ranks. where significant were found, related - samples wilcoxon signed rank tests ensued for determination of where the differences lie. analyses were performed using spss 18.0 for windows (spss, chicago, il). twelve (10 male and 2 female) nonobese (bmi 25.3 3.0 kg / m), physically active (vo2max 51.2 10.8 ml kg min) individuals aged 1762 years (mean age 31.8 15.3 years) took part in the study. mean diabetes duration was 12.5 10.0 years, and participants were in moderate to good control of their blood glucose levels (hba1c 7.1 1.1%). five participants were receiving insulin by mdi, while seven were using continuous subcutaneous insulin infusion. 1. information regarding treadmill speeds / inclines as well as the workloads for the resistance exercise sessions is provided in supplementary table 1. a significant interaction between time and exercise modality was observed (p < 0.001) for mean exercise glucose levels indicating that the total declines and the rates of decline in plasma glucose levels differed among sessions (fig . 1). there were no significant differences among sessions in pre - exercise baseline plasma glucose concentration. a gradual decline in plasma glucose concentration occurred with resistance exercise (from 8.4 2.7 to 6.8 2.3 mmol / l over the 45-min session), ing in levels that were significantly lower than baseline by the end of exercise (p = 0.008). no changes from baseline were detected throughout the first 45 min of the no - exercise session (from 8.4 3.5 to 8.6 3.8 mmol / l). in contrast, during the aerobic exercise, plasma glucose levels declined rapidly and more dramatically (from 9.2 3.4 to 5.8 2.0 mmol / l over 45 min), ing in significant changes from baseline within 10 min. glucose levels in the aerobic session were lower than the no - exercise session after 30 min of the activity. mean se plasma glucose during the experimental sessions (represented by box) and 60 min of recovery (n = 12 for aerobic exercise and no - exercise control ; n = 11 for resistance exercise). , no - exercise control; , resistance exercise, , aerobic exercise. differences were only considered statistically significant if still significant after bonferroni corrections for multiple comparisons. during exercise, participants were provided with glucose tablets if blood glucose fell to < 4.5 mmol / l. a significant interaction of time and exercise modality was also observed in mean plasma glucose levels during recovery (p < 0.001). plasma glucose levels were stable after the resistance exercise and no - exercise sessions but increased by 2.2 0.6 plasma glucose levels were not different from either no - exercise or resistance exercise at 60 min postexercise. the number of participants requiring glucose tablets during the testing session were two, nine, and three for the no - exercise control, aerobic, and resistance exercise sessions, respectively (supplementary table 2). differences were significant between no - exercise control and aerobic exercise (p = 0.007). there were no significant differences in carbohydrate intake among conditions on the day before or the day after the laboratory session or in the 6 h after exercise (table 1); however, carbohydrate intake was higher on the exercise testing day in the aerobic exercise session compared with the resistance exercise session (p = 0.013), mostly because of differences in supplementation during exercise. two participants using insulin pumps chose to omit their usual insulin bolus with the glucerna bar before exercise, and one insisted on suspending basal insulin (instead of a 50% reduction) when learning upon arrival at the laboratory that it was the day for aerobic activity. daily insulin intake did not differ significantly among conditions on any day of sensor wear. insulin and carbohydrate intake during the 6 h after exercise * pearson correlations between capillary glucose levels measured on handheld meters and interstitial glucose levels measured by cgm were 0.95, 0.90, and 0.94 during nonlaboratory periods in the resistance exercise, aerobic, and no - exercise control sessions, respectively. during the 24 h before either exercise trial or no - exercise control , there were no significant differences among sessions in the total time spent in hypoglycemia, auc for hypoglycemia, number of hyperglycemic events, time spent in a hyperglycemic state, auc for hyperglycemia, or mean blood glucose. postexercise cgm data were only available for 11 and 10 of 12 participants in the no - exercise and aerobic exercise sessions, respectively, because of equipment malfunction in the remaining three sessions. data were available for all 12 participants in the resistance exercise session. in total, there were 124 paired handheld meter and cgm values for the no - exercise control condition, 113 for the aerobic condition, and 115 for the resistance exercise condition. a marginal effect of time (p = 0.073) was found in the analysis of the cgm data from 1 to 6 h postexercise. higher mean interstitial glucose concentrations were found in the fourth and fifth hours after the aerobic exercise session compared with the resistance exercise session (p = 0.018 at 5 h postexercise) (fig . , no - exercise control session ; , aerobic exercise session ; , resistance exercise session . the box represents the period of time where glucose was significantly higher after aerobic exercise compared with resistance exercise ( p < 0.05). n = 11 (no - exercise control), n = 10 (aerobic), and n = 12 (resistance). although there were twice as many nocturnal hypoglycemic excursions (table 2) detected by cgm devices after resistance exercise (nine in total) versus aerobic exercise and no exercise (four for each), differences among conditions were not statistically significant. there was, however, a trend of more episodes of nocturnal hyperglycemia after resistance exercise (p = 0.059) compared with the pre - exercise night, but differences in mean glucose levels were not significant. summary of overnight cgm data for the night after resistance exercise, aerobic exercise, and no - exercise control 1. information regarding treadmill speeds / inclines as well as the workloads for the resistance exercise sessions is provided in supplementary table 1. a significant interaction between time and exercise modality was observed (p < 0.001) for mean exercise glucose levels indicating that the total declines and the rates of decline in plasma glucose levels differed among sessions (fig . 1). there were no significant differences among sessions in pre - exercise baseline plasma glucose concentration. a gradual decline in plasma glucose concentration occurred with resistance exercise (from 8.4 2.7 to 6.8 2.3 mmol / l over the 45-min session), ing in levels that were significantly lower than baseline by the end of exercise (p = 0.008). no changes from baseline were detected throughout the first 45 min of the no - exercise session (from 8.4 3.5 to 8.6 3.8 mmol / l). in contrast, during the aerobic exercise, plasma glucose levels declined rapidly and more dramatically (from 9.2 3.4 to 5.8 2.0 mmol / l over 45 min), ing in significant changes from baseline within 10 min. glucose levels in the aerobic session were lower than the no - exercise session after 30 min of the activity. mean se plasma glucose during the experimental sessions (represented by box) and 60 min of recovery (n = 12 for aerobic exercise and no - exercise control ; n = 11 for resistance exercise). , no - exercise control; , resistance exercise, , aerobic exercise. differences were only considered statistically significant if still significant after bonferroni corrections for multiple comparisons. during exercise, participants were provided with glucose tablets if blood glucose fell to < 4.5 mmol / l. a significant interaction of time and exercise modality was also observed in mean plasma glucose levels during recovery (p < 0.001). plasma glucose levels were stable after the resistance exercise and no - exercise sessions but increased by 2.2 0.6 plasma glucose levels were not different from either no - exercise or resistance exercise at 60 min postexercise. 1. information regarding treadmill speeds / inclines as well as the workloads for the resistance exercise sessions is provided in supplementary table 1. a significant interaction between time and exercise modality was observed (p < 0.001) for mean exercise glucose levels indicating that the total declines and the rates of decline in plasma glucose levels differed among sessions (fig . 1). there were no significant differences among sessions in pre - exercise baseline plasma glucose concentration. a gradual decline in plasma glucose concentration occurred with resistance exercise (from 8.4 2.7 to 6.8 2.3 mmol / l over the 45-min session), ing in levels that were significantly lower than baseline by the end of exercise (p = 0.008). no changes from baseline were detected throughout the first 45 min of the no - exercise session (from 8.4 3.5 to 8.6 3.8 mmol / l). in contrast, during the aerobic exercise, plasma glucose levels declined rapidly and more dramatically (from 9.2 3.4 to 5.8 2.0 mmol / l over 45 min), ing in significant changes from baseline within 10 min. glucose levels in the aerobic session were lower than the no - exercise session after 30 min of the activity. mean se plasma glucose during the experimental sessions (represented by box) and 60 min of recovery (n = 12 for aerobic exercise and no - exercise control ; n = 11 for resistance exercise). , no - exercise control; , resistance exercise, , aerobic exercise. differences were only considered statistically significant if still significant after bonferroni corrections for multiple comparisons. during exercise, participants were provided with glucose tablets if blood glucose fell to < 4.5 mmol / l. a significant interaction of time and exercise modality was also observed in mean plasma glucose levels during recovery (p < 0.001). plasma glucose levels were stable after the resistance exercise and no - exercise sessions but increased by 2.2 0.6 plasma glucose levels were not different from either no - exercise or resistance exercise at 60 min postexercise. the number of participants requiring glucose tablets during the testing session were two, nine, and three for the no - exercise control, aerobic, and resistance exercise sessions, respectively (supplementary table 2). differences were significant between no - exercise control and aerobic exercise (p = 0.007). there were no significant differences in carbohydrate intake among conditions on the day before or the day after the laboratory session or in the 6 h after exercise (table 1); however, carbohydrate intake was higher on the exercise testing day in the aerobic exercise session compared with the resistance exercise session (p = 0.013), mostly because of differences in supplementation during exercise. two participants using insulin pumps chose to omit their usual insulin bolus with the glucerna bar before exercise, and one insisted on suspending basal insulin (instead of a 50% reduction) when learning upon arrival at the laboratory that it was the day for aerobic activity. daily insulin intake did not differ significantly among conditions on any day of sensor wear. pearson correlations between capillary glucose levels measured on handheld meters and interstitial glucose levels measured by cgm were 0.95, 0.90, and 0.94 during nonlaboratory periods in the resistance exercise, aerobic, and no - exercise control sessions, respectively. during the 24 h before either exercise trial or no - exercise control, there were no significant differences among sessions in the total time spent in hypoglycemia, auc for hypoglycemia, number of hyperglycemic events, time spent in a hyperglycemic state, auc for hyperglycemia, or mean blood glucose. postexercise cgm data were only available for 11 and 10 of 12 participants in the no - exercise and aerobic exercise sessions, respectively, because of equipment malfunction in the remaining three sessions. , there were 124 paired handheld meter and cgm values for the no - exercise control condition, 113 for the aerobic condition, and 115 for the resistance exercise condition. a marginal effect of time (p = 0.073) was found in the analysis of the cgm data from 1 to 6 h postexercise. higher mean interstitial glucose concentrations were found in the fourth and fifth hours after the aerobic exercise session compared with the resistance exercise session (p = 0.018 at 5 h postexercise) (fig . 2). mean se glucose as measured by cgm from 1 to 12 h postexercise. , no - exercise control session; , aerobic exercise session; , resistance exercise session. the box represents the period of time where glucose was significantly higher after aerobic exercise compared with resistance exercise (p < 0.05). n = 11 (no - exercise control), n = 10 (aerobic), and n = 12 (resistance). although there were twice as many nocturnal hypoglycemic excursions (table 2) detected by cgm devices after resistance exercise (nine in total) versus aerobic exercise and no exercise (four for each), differences among conditions were not statistically significant. there was, however, a trend of more episodes of nocturnal hyperglycemia after resistance exercise (p = 0.059) compared with the pre - exercise night, but differences in mean glucose levels were not significant. summary of overnight cgm data for the night after resistance exercise, aerobic exercise, and no - exercise control resistance exercise ed in much smaller declines in blood glucose during exercise than aerobic exercise or no exercise in individuals with type 1 diabetes. less carbohydrate supplementation was required during resistance exercise versus aerobic exercise, which would have attenuated some of the hypoglycemic effects of the aerobic activity. in contrast to resistance exercise and no exercise, aerobic exercise was associated with greater increases in glucose levels during early recovery, which ed in a trend toward higher glucose concentrations in late recovery (as measured by cgm 36 h postexercise). these trends were observed in the absence of any significant differences in insulin dosage or carbohydrate intake during this time. mean blood glucose levels after resistance exercise were similar to those when no exercise was performed: more stable during early recovery and within a healthier range (56 mmol / l) during late recovery. as such, performance of resistance exercise may represent an alternative strategy to prevent the acute decline in blood glucose levels observed with aerobic exercise while maintaining more favorable postexercise glucose levels. there was, however, a tendency toward more frequent, albeit mild, nocturnal hypoglycemia after resistance exercise sessions, which deserves further scrutiny. the mechanisms for the more dramatic reduction in blood glucose levels during aerobic versus resistance exercise are unclear, but the reliance on anaerobic sources of fuel production during resistance exercise rather than aerobic sources (i.e., less reliance on blood glucose) may have played a role. previous studies involving anaerobic activity in individuals with type 1 diabetes (intermittent 4-s sprints or a 10-s sprint pre- or postexercise) found slower declines in blood glucose concentration during exercise and smaller decreases in postexercise glucose concentrations in comparison with low - intensity aerobic exercise alone. insulin and cortisol levels were comparable across conditions in these studies and were therefore unlikely to be responsible for the differential patterns of blood glucose response. growth hormone and catecholamines, meanwhile, were elevated after sprinting, potentially enhancing lipolysis and glycogenolysis, respectively, thereby potentially stabilizing blood glucose levels. it is undetermined whether these hormones are responsible for stabilizing blood glucose levels after resistance exercise in individuals with type 1 diabetes; however, both growth hormone and catecholamines are known to increase significantly in individuals without diabetes during resistance exercise protocols similar to the one used in the current study. attenuated declines in blood glucose concentration may also be related to increased lactate production during resistance exercise. in comparing the hormonal responses to various resistance exercise protocols, smilios et al. found that two sets of 10 repetitions of chest press, lateral pull down, and squat (a stimulus of smaller magnitude than the one used in the current study) ed in a fourfold increase in blood lactate levels, with elevated lactate persisting for at least 30 min postexercise in individuals without diabetes. while we are unaware of published data on lactate production during resistance exercise in individuals with type 1 diabetes, there is no reason to believe that lactate production would be impaired in this population. indeed, other anaerobic activity (high - intensity cycling) produced elevated lactate levels persisting up to 30 min postexercise in individuals with type 1 diabetes. we did not measure lactate in the current study but can surmise that blood lactate levels would have increased more during resistance exercise where glycolysis predominates than during aerobic exercise where lipolysis generates much of the energy required, especially in physically fit individuals. overall, there were no significant differences among the conditions with respect to any measures of hypoglycemia or mean nocturnal blood glucose levels (table 2), although resistance exercise was associated with a nonsignificant trend for more nocturnal hypoglycemia. while we are unaware of any study examining nocturnal blood glucose levels after resistance exercise in type 1 diabetic subjects, mcmahon et al. found that adolescents with type 1 diabetes had a higher glucose infusion requirement to maintain euglycemia between midnight and 4:00 a.m. after performing evening aerobic exercise than if no exercise had been performed. this coincides with the time when the lowest nocturnal glucose levels were found after both exercise sessions in our study (fig . 2), although differences among conditions were not significant. as mcmahon et al. surmised that delayed increases in postexercise glucose needs relate to replenishment of glycogen stores, a higher frequency of low blood glucose after resistance exercise (which relies more on glycogen for fuel) might be expected. it is also possible that differences in food and insulin intake (table 2), while not statistically significant, could have had a minor effect on postexercise glucose profiles. in addition, while participants were asked to match their food and insulin intake both pre- and postexercise as closely as possible among the sessions, some differences may not have been reported. this does not, however, detract from the findings, as patient decisions regarding insulin dosage and carbohydrate intake play an essential role in diabetes management. as there is currently very little information available with respect to insulin adjustments for resistance exercise, participants in the current study were relying to a great extent on personal experience and judgment. higher physical activity levels in individuals with type 1 diabetes have been associated with lower frequency and severity of diabetes complications; however, fear of hypoglycemia is generally the strongest barrier to physical activity for this population. resistance exercise is associated with improvements in muscular strength, improved lipid profiles, lower insulin needs, and lower self - monitored blood glucose levels in individuals with type 1 diabetes. it also carries many of the same benefits as aerobic exercise (higher bone mineral density, increased insulin sensitivity, and improved cardiovascular function) and may therefore be a safe and effective option for this population. interestingly, we observed more exercise - associated glycemic fluctuation with aerobic exercise compared with resistance exercise. during the activity, aerobic exercise was associated with greater reductions in glycemia, while in early recovery there was more rebound hyperglycemia compared with resistance exercise. thus, one could conclude that resistance exercise may be more beneficial as far as glucose stability is concerned. moreover, as individuals with type 1 diabetes may also have an increased risk of myopathy and complications associated with insulin resistance, performing regular resistance exercise may help maintain or improve muscle mass and metabolism. meanwhile, it should also be noted that postexercise hypoglycemia might occur more frequently in individuals who have changed their exercise routine to incorporate resistance training or for patients unaccustomed to exercise. in summary , our findings suggest that in trained individuals with type 1 diabetes who habitually practice both aerobic and resistance exercise, resistance exercise may in more stable glucose levels both during and after exercise than aerobic exercise, which may explain the beneficial effects on hba1c found in previous intervention studies involving resistance exercise. the trend toward more frequent, albeit mild, nocturnal hypoglycemia after resistance exercise reported in our study, however, indicates the possible need to develop more effective clinical management protocols for different forms of exercise.

objectivein type 1 diabetes, small studies have found that resistance exercise (weight lifting) reduces hba1c. in the current study, we examined the acute impacts of resistance exercise on glycemia during exercise and in the subsequent 24 h compared with aerobic exercise and no exercise.research design and methodstwelve physically active individuals with type 1 diabetes (hba1c 7.1 1.0%) performed 45 min of resistance exercise (three sets of seven exercises at eight repetitions maximum), 45 min of aerobic exercise (running at 60% of vo2max), or no exercise on separate days. plasma glucose was measured during and for 60 min after exercise. interstitial glucose was measured by continuous glucose monitoring 24 h before, during, and 24 h after exercise.treatment-by-time interactions (p < 0.001) were found for changes in plasma glucose during and after exercise. plasma glucose decreased from 8.4 2.7 to 6.8 2.3 mmol / l (p = 0.008) during resistance exercise and from 9.2 3.4 to 5.8 2.0 mmol / l (p = 0.001) during aerobic exercise. no significant changes were seen during the no - exercise control session. during recovery, glucose levels did not change significantly after resistance exercise but increased by 2.2 0.6 mmol / l (p = 0.023) after aerobic exercise. mean interstitial glucose from 4.5 to 6.0 h postexercise was significantly lower after resistance exercise versus aerobic exercise.resistance exercise causes less initial decline in blood glucose during the activity but is associated with more prolonged reductions in postexercise glycemia than aerobic exercise. this might account for hba1c reductions found in studies of resistance exercise but not aerobic exercise in type 1 diabetes.

stomata play a major role in water loss and carbon gain by regulating diffusive conductance in leaves. it is widely accepted that stomata respond to perturbations by many soil - plant - atmosphere signals but there is little agreement regarding the mechanism(s) by which they sense and react to such perturbations (buckley, 2005). the phenomenon of autonomous, cyclic opening and closing movements of stomata within a period of less than 3 h was observed over 40 years ago (barrs, 1971). those measurements showed that when the periodic variations in transpiration are plotted against time, a sine wave with a period of 1090 min is obtained (barrs and klepper, 1968 ; cox, 1968 ; barrs, 1971 ; teoh and palmer, 1971). such movements are most readily detected in a stable environment, but there seems to be no a priori reason why such movements can not persist, albeit with some modifications, in non - stable environments (barrs, 1971). traditionally, two approaches have been taken to study the effect of environmental conditions on oscillatory transpiration. the first involves tracking the variations in transpiration rate of a small part of, or a whole leaf. the second focuses on the behaviour of individual stomata. adopting the first approach, ehrler et al. , naidoo and von willert, herppich and von willert, jarvis et al. studied oscillations in whole leaves from a large variety of species, most often using gas - exchange techniques. measurements of chlorophyll a fluorescence (siebke and weis, 1995), leaf temperature or leaf water potential in terms of water relations (ehrler et al ., 1965 ; barrs and klepper, 1968 ; lang et al ., 1969 ; mcburney and costigan, 1984 ; naidoo and von willert, 1994 ; herppich and von willert, 1995 ; prytz et al ., 2003) have also been used to record stomatal oscillations. a common in all of these studies was that in - phase oscillations are induced by various agents, bringing about abrupt stomatal opening or closure. reports of the synchronous rhythmic pattern of stomatal activity in different leaves on the same plant were reviewed by hopmans and lang et al. these authors found oscillations in leaf - xylem water potential and proposed that these are a necessary condition for the stomata of all leaves to oscillate in phase. oscillations in root - xylem pressure were measured by wegner and zimmermann and prytz et al. , who noted that the synchronous behaviour observed during oscillatory transpiration indicates strong coupling among stomata; however, they did not suggest a coupling agent. herppich and von willert measured pronounced oscillations in stomatal apertures under certain climatic conditions and noted that changes in transpirational water loss during these oscillations are closely accompanied by changes in leaf water potential (l). on a stomatal scale, the simultaneous existence of different stomatal apertures, extents of stomatal opening across a leaf, and the possible implications of cross - co2 exchange were first discussed by laisk and co - workers (laisk et al . terashima argued that under certain physiological conditions, the spatial distribution of different stomatal conductances is non - random (patchy), i.e. certain areas of a leaf can have a very different conductance from others. patchy stomatal closure has been observed, particularly under water stress (downton et al ., 1988 ; beyschlag et al ., 1992) and low humidity (mott and parkhurst, 1991 ; mott et al ., 1993, 1999), and following abscisic acid application (daley et al ., 1989 ; terashima, 1992 ; mott, 1995). several models of the mechanism responsible for the oscillations in stomata aperture have been described (lang et al ., 1969 ; , 1997 ; mcainsh et al ., 1997 ; shabala et al ., 1997 ; meleshchenko, 2000 ; bohn et al ., 2001), presenting a common basic assumption: that the oscillation behaviour is part of a feedback loop in which evaporation rate influences the stomatal aperture which, in turn, affects evaporation rate. some have suggested that stomatal cycling is induced by internal fluctuations in water potential and flow resistance within the plant (steudle, 2000). despite the fact that oscillations have been intensively studied in single stomata, leaf patches and whole leaves, oscillations on a whole - plant scale have been rather neglected. the studies of rose and co - authors (rose and rose, 1994 ; rose et al ., 1994) and steppe et al. are, to the best of our knowledge, the only ones in which oscillations in whole - plant transpiration (wpt) have been measured. a recent study, which revealed that the long - distance signal triggering the reduction in leaf conductance under water stress is hydraulic rather than root - produced abscisic acid (christmann et al ., 2007), led us to hypothesize that synchronized stomatal activity at the whole - plant level could be the of a hydraulic signal spreading rapidly via the plant's vascular system. consequently, this study aimed to isolate and characterize the autonomous, self - regulated oscillations in wpt rate that develop with increasing water stress. the experimental study was conducted in greenhouses at the faculty of agriculture, food and environment in rehovot, israel. the experimental set - up included 3.9 l growing pots placed on temperature - compensated load cells (tadea - huntleigh, natanya, israel) connected to a cr10 data logger (campbell scientific inc ., logan, ut). the pots were filled with a commercial growing medium (a mixture of peat and tuff scoria) and each contained one plant. each pot was immersed in a plastic container (1321.531.5 cm, hwl) through a hole in its top cover. the water level fell to 2 cm above the pot base after the irrigation events due to a drainage hole in the container side wall. the pot - container system ensured that water was available to the plant throughout the day following irrigation, without supplemental irrigation. a commercial fertilizer solution (super grow 6 - 6 - 6 + 3 hortical, kadima, israel) was added at 0.2% (v / v) daily with the irrigation water (fertigation). this set - up ensured that (i) the plants would not be subjected to water stress throughout the following day, and (ii) the container weight on the following day would decrease monotonically solely due to plant transpiration. ailsa craig ) were grown in a controlled - environment greenhouse, with the temperature set at 18 c during the night and 35 c during the midday hours with periods of gradual variation between them. the effect of water stress on the patterns of wpt - rate oscillations was studied in a dehydration experiment using the tomato plants (at the age of 1012 weeks). the plants were dehydrated by stopping irrigation, removing the containers in which the pots were immersed and allowing the plant to deplete the water from the growth medium in the pot for five successive days. average and momentary changes in atmospheric demand were assessed by measuring the weight loss from a vertical woven floor rag (0.14 m), the lower part of which was submerged in water in a container (noted hereafter as wet wick) that was placed on a load cell. to isolate intrinsic noise associated with the measuring and data - acquisition systems from the short - term fluctuations in plant transpiration, a constant load of 6 kg (typical of the container+pot+plant weight) was placed on the load cells in the greenhouse for several days, during which the weight variation was monitored. the data from the load cells with plants, wet wick and constant load were analysed by the following time - series analysis. weight readings were taken every 10 s. the load - cell reading stabilized after 2 s following stimulation by dropping a 70-g steel ball from a height of 700 mm (manufacturer 's data). thus, a 10 s weight - sampling interval ensured that the maximum rate of weight decrease (0.5 g per 10 s) was appropriately followed. the data analysis consisted of two steps: the first included averaging 10 s readings over 3 min periods. this stage was aimed at decreasing the number of data points in further data analysis (second stage). the 3 min periods are clearly lower than the oscillation frequency (2040 min) and higher than the nyquist frequency (the highest frequency at which meaningful information can be obtained from a set of data). leaves were excised from each plant separately and their areas were measured using a leaf area scanner (licor 3210, lincoln, ne). the rate of water loss from the system, being the negative value of the wpt rate, was calculated by the first derivative of the measured weight time serieswhere wk and wk+1 are, respectively, the measured weights at time tk and the following time step tk+1 in general, differentiation acts as a high - pass filter, thereby significantly amplifying the high - frequency noise, and noises are amplified as the measurement (sampling) interval (tk+1tk, equation 1) decreases. consequently, a signal treatment is essential when the noise associated with the transpiration - induced weight decrease is differentiated together with the high - frequency noises associated with the load - cell and data - acquisition systems, as both are embedded in the measured time series, owing to amplification of the noise introduced by the latter. in fact, measurement errors, which can never be avoided, complicate the differentiation, because these amplify the noise to such an extent that additional signal treatment is essential. the noise associated with the differentiation of noisy signals has stimulated a large number of investigations which have led to several solutions, in both the time and frequency domains (savitzky and golay, 1964 ; cullum, 1971 ; anderssen and bloomfield, 1974a, b ; wahba, 1975 ; rice and rosenblatt, 1983 ; scott and scott, 1989). noise can be reduced or eliminated by smoothing (detrending) the measured data (time series) so that the smoothed data becomes stationary prior to the subsequent stage of spectral analysis. many methods are used for smoothing noisy data in time - series analyses (harvey and shephard, 1993). these methods can be categorized as non - parametric smoothing and non - parametric regression (fitting polynomials of various orders, exponential functions, symmetrical and asymmetrical - transition functions, etc to the measured data]. smoothing should be performed with care since substantially different can be obtained after differentiation, despite the high r values that are usually obtained for the different smoothing methods. the savitzky and golay method (s - g) was used in this study to smooth the measured time series. this smoothing method is based on least - squares quadratic polynomial fitting (although higher orders can also be used) across a moving window within the data (press et al . the s - g method can be applied for various breadths of filtering windows, and it is considered a very good way of producing accurate smooth derivatives . the fit improves and includes fluctuations of higher frequencies and amplitudes with decreasing breadth of the filtering window . s - g with 30 data points was ultimately chosen for the window breadth as its time derivative provided a smooth ( but not too smooth) pattern. it is assumed herein that the container weight - time series follows an additive modelwhere w is the value that the weight at time tk would have if it varied smoothly with time, and k is the deviation from that value. the system's weight oscillations superimposed on the smoothed time series also constitute a time series, designated residual time series (residuals are the differences between the measured data and the fitted curve). when the mean of the residual time series is zero, the residual time series k (equation 2) is a superposition of two time series: one made up of residuals that originate from the noises related to data acquisition, and the other of residuals originating from either the ambient conditions and/or the intrinsic oscillations in wpt. the time series for the constant - weight, wet - wick, and whole - plant runs (which were independently measured) were used to examine its randomness (white noise). the randomness of the noise originated from the data - acquisition system and the repeating patterns (periodicity) in the wet - wick and plant oscillations were examined by the autocorrelation function. this is a mathematical tool for finding repeating patterns, such as the presence of a periodic signal which has been buried under the noise function, by expressing the similarity between observations as a function of the time separation between them. if the noise is a white noise, the autocorrelation function should be near zero for any and all time - lag separations. if not a white noise, then one or more (except at lag=0) of the autocorrelations will be significantly non - zero. the spectrum analysis of k was used to explore the existence of cyclical patterns by decomposing the complex time series with cyclical components into a few underlying sinusoidal (sine and cosine) functions of particular wavelengths. the characteristics of the phenomenon of interest become apparent when the important underlying cyclical components are identified. the spectrum analysis enabled us to uncover a few cycles of different lengths in the time series of interest, which at first looked more or less like random noise. the spectrum (amplitudes versus frequencies) of the residual time series was calculated by fast fourier transform (fft), which decomposes a time - domain signal or time series into complex exponentials (sines and cosines). the spectrum of the constant - load residual time series was used to determine the frequency threshold that would be used to filter out the high - frequency noise (low - pass filter) from the plant - weight residual time series. subsequently, the filtered spectrum was reconstructed back to a time series (in the time domain) by inverse fft. the calculations of the above were executed by tablecurve 2d (systat software inc, ca). the time derivative of the reconstructed low - pass - filtered time series d(k)/dt (( k) is the low - pass filter of k ) provided the oscillatory transpiration rate that superimposes the smoothed wpt rate. the evaporation rate (smoothed and oscillatory) from the wet wick integrates the effect of the ambient conditions on both the wick and the plant. a high correlation between the two time series is expected under optimal growth conditions, while deviations are likely to occur when the plants are exposed to stresses. the wet - wick evaporation rate - time series is regarded as the input to the plant system and the wpt rate - time series as the output. hence, a comparison between the patterns of smoothed and low - pass oscillations in wpt rate and wet - wick evaporation rate can reveal whether the synchronized oscillations in wpt rate are mainly driven by fluctuations in ambient conditions in the greenhouse, or if they tend to become autonomous under certain independent conditions (e.g. water stress). the relationship between the two time series can be interpreted by quantitative and qualitative means. a qualitative estimation of the correlation between the transpiration- and evaporation - rate time series can be obtained by plotting the two time series on the same plot and a quantitative estimation is obtained by the cross - correlation function. a plot of the time series enables isolating periods throughout the day during which the two time series deviate, and relating these periods to the variation of the smoothed patterns of these variables or to other independent variables in the system. a cross - correlation function with a single and pronounced peak indicates that the transpiration and wet - wick evaporation oscillatory patterns are related, and that the oscillatory wpt rate is driven by the fluctuations in ambient conditions (evaporative demand). a peak value close to 1 located at lag=0 indicates that the changes in wpt rate are fully and instantaneously driven by the fluctuations in evaporative demand, as reflected by the wet wick. as the wpt rate of a well - watered plant responds to environmental conditions, a good correlation coefficient is expected between well - watered plants and the wet wick, the latter closely simulating the evaporative demand. however, a perfect correlation coefficient was not anticipated as the boundary - layer resistances between the wet wick and the plant crown are dissimilar, the thermal properties and heat capacity of the two systems differ, and the plant can control its diffusive resistance by changing stomatal aperture. on the other hand, a rippled, low - value cross - correlation function, with no clear peak, can be interpreted as a lack of dependence between the two time series, namely, the oscillations in wpt rate are autonomous and self - regulated, independent of the fluctuations in ambient conditions. the experimental study was conducted in greenhouses at the faculty of agriculture, food and environment in rehovot, israel. the experimental set - up included 3.9 l growing pots placed on temperature - compensated load cells (tadea - huntleigh, natanya, israel) connected to a cr10 data logger (campbell scientific inc ., logan, ut). the pots were filled with a commercial growing medium (a mixture of peat and tuff scoria) and each contained one plant. each pot was immersed in a plastic container (1321.531.5 cm, hwl) through a hole in its top cover. the water level fell to 2 cm above the pot base after the irrigation events due to a drainage hole in the container side wall. the pot - container system ensured that water was available to the plant throughout the day following irrigation, without supplemental irrigation. a commercial fertilizer solution (super grow 6 - 6 - 6 + 3 hortical, kadima, israel) was added at 0.2% (v / v) daily with the irrigation water (fertigation). this set - up ensured that (i) the plants would not be subjected to water stress throughout the following day, and (ii) the container weight on the following day would decrease monotonically solely due to plant transpiration. ailsa craig ) were grown in a controlled - environment greenhouse, with the temperature set at 18 c during the night and 35 c during the midday hours with periods of gradual variation between them. the effect of water stress on the patterns of wpt - rate oscillations was studied in a dehydration experiment using the tomato plants (at the age of 1012 weeks). the plants were dehydrated by stopping irrigation, removing the containers in which the pots were immersed and allowing the plant to deplete the water from the growth medium in the pot for five successive days. average and momentary changes in atmospheric demand were assessed by measuring the weight loss from a vertical woven floor rag (0.14 m), the lower part of which was submerged in water in a container (noted hereafter as wet wick) that was placed on a load cell. to isolate intrinsic noise associated with the measuring and data - acquisition systems from the short - term fluctuations in plant transpiration, a constant load of 6 kg (typical of the container+pot+plant weight) was placed on the load cells in the greenhouse for several days, during which the weight variation was monitored. the data from the load cells with plants, wet wick and constant load were analysed by the following time - series analysis. weight readings were taken every 10 s. the load - cell reading stabilized after 2 s following stimulation by dropping a 70-g steel ball from a height of 700 mm (manufacturer 's data). thus, a 10 s weight - sampling interval ensured that the maximum rate of weight decrease (0.5 g per 10 s) was appropriately followed. the data analysis consisted of two steps: the first included averaging 10 s readings over 3 min periods. this stage was aimed at decreasing the number of data points in further data analysis (second stage). the 3 min periods are clearly lower than the oscillation frequency (2040 min) and higher than the nyquist frequency (the highest frequency at which meaningful information can be obtained from a set of data). leaves were excised from each plant separately and their areas were measured using a leaf area scanner (licor 3210, lincoln, ne). the rate of water loss from the system, being the negative value of the wpt rate, was calculated by the first derivative of the measured weight time serieswhere wk and wk+1 are, respectively, the measured weights at time tk and the following time step tk+1 in general, differentiation acts as a high - pass filter, thereby significantly amplifying the high - frequency noise, and noises are amplified as the measurement (sampling) interval (tk+1tk, equation 1) decreases. consequently, a signal treatment is essential when the noise associated with the transpiration - induced weight decrease is differentiated together with the high - frequency noises associated with the load - cell and data - acquisition systems, as both are embedded in the measured time series, owing to amplification of the noise introduced by the latter. in fact, measurement errors, which can never be avoided, complicate the differentiation, because these amplify the noise to such an extent that additional signal treatment is essential. the noise associated with the differentiation of noisy signals has stimulated a large number of investigations which have led to several solutions, in both the time and frequency domains (savitzky and golay, 1964 ; cullum, 1971 ; anderssen and bloomfield, 1974a, b ; wahba, 1975 ; rice and rosenblatt, 1983 ; scott and scott, 1989). noise can be reduced or eliminated by smoothing (detrending) the measured data (time series) so that the smoothed data becomes stationary prior to the subsequent stage of spectral analysis. many methods are used for smoothing noisy data in time - series analyses (harvey and shephard, 1993). these methods can be categorized as non - parametric smoothing and non - parametric regression (fitting polynomials of various orders, exponential functions, symmetrical and asymmetrical - transition functions, etc to the measured data]. smoothing should be performed with care since substantially different can be obtained after differentiation, despite the high r values that are usually obtained for the different smoothing methods. the savitzky and golay method (s - g) was used in this study to smooth the measured time series. this smoothing method is based on least - squares quadratic polynomial fitting (although higher orders can also be used) across a moving window within the data (press et al ., 1988). the s - g method can be applied for various breadths of filtering windows, and it is considered a very good way of producing accurate smooth derivatives. the fit improves and includes fluctuations of higher frequencies and amplitudes with decreasing breadth of the filtering window. s - g with 30 data points was ultimately chosen for the window breadth as its time derivative provided a smooth (but not too smooth) pattern. it is assumed herein that the container weight - time series follows an additive modelwhere w is the value that the weight at time tk would have if it varied smoothly with time, and k is the deviation from that value. the system's weight oscillations superimposed on the smoothed time series also constitute a time series, designated residual time series (residuals are the differences between the measured data and the fitted curve). when the mean of the residual time series is zero, the trend of the measured time series has been properly removed. the residual time series k (equation 2) is a superposition of two time series: one made up of residuals that originate from the noises related to data acquisition, and the other of residuals originating from either the ambient conditions and/or the intrinsic oscillations in wpt. the time series for the constant - weight, wet - wick, and whole - plant runs (which were independently measured) were used to examine its randomness (white noise). the randomness of the noise originated from the data - acquisition system and the repeating patterns (periodicity) in the wet - wick and plant oscillations were examined by the autocorrelation function. this is a mathematical tool for finding repeating patterns, such as the presence of a periodic signal which has been buried under the noise function, by expressing the similarity between observations as a function of the time separation between them. if the noise is a white noise, the autocorrelation function should be near zero for any and all time - lag separations. if not a white noise, then one or more (except at lag=0) of the autocorrelations will be significantly non - zero. the spectrum analysis of k was used to explore the existence of cyclical patterns by decomposing the complex time series with cyclical components into a few underlying sinusoidal (sine and cosine) functions of particular wavelengths. the characteristics of the phenomenon of interest become apparent when the important underlying cyclical components are identified. the spectrum analysis enabled us to uncover a few cycles of different lengths in the time series of interest, which at first looked more or less like random noise. the spectrum (amplitudes versus frequencies) of the residual time series was calculated by fast fourier transform (fft), which decomposes a time - domain signal or time series into complex exponentials (sines and cosines). the spectrum of the constant - load residual time series was used to determine the frequency threshold that would be used to filter out the high - frequency noise (low - pass filter) from the plant - weight residual time series. subsequently, the filtered spectrum was reconstructed back to a time series (in the time domain) by inverse fft. the calculations of the above were executed by tablecurve 2d (systat software inc, ca). the time derivative of the reconstructed low - pass - filtered time series d(k)/dt (( k) is the low - pass filter of k ) provided the oscillatory transpiration rate that superimposes the smoothed wpt rate. the evaporation rate (smoothed and oscillatory) from the wet wick integrates the effect of the ambient conditions on both the wick and the plant. a high correlation between the two time series is expected under optimal growth conditions, while deviations are likely to occur when the plants are exposed to stresses. the wet - wick evaporation rate - time series is regarded as the input to the plant system and the wpt rate - time series as the output. hence, a comparison between the patterns of smoothed and low - pass oscillations in wpt rate and wet - wick evaporation rate can reveal whether the synchronized oscillations in wpt rate are mainly driven by fluctuations in ambient conditions in the greenhouse, or if they tend to become autonomous under certain independent conditions (e.g. water stress). the relationship between the two time series can be interpreted by quantitative and qualitative means. a qualitative estimation of the correlation between the transpiration- and evaporation - rate time series can be obtained by plotting the two time series on the same plot and a quantitative estimation is obtained by the cross - correlation function. a plot of the time series enables isolating periods throughout the day during which the two time series deviate, and relating these periods to the variation of the smoothed patterns of these variables or to other independent variables in the system. a cross - correlation function with a single and pronounced peak indicates that the transpiration and wet - wick evaporation oscillatory patterns are related, and that the oscillatory wpt rate is driven by the fluctuations in ambient conditions (evaporative demand). a peak value close to 1 located at lag=0 indicates that the changes in wpt rate are fully and instantaneously driven by the fluctuations in evaporative demand, as reflected by the wet wick. as the wpt rate of a well - watered plant responds to environmental conditions, a good correlation coefficient is expected between well - watered plants and the wet wick, the latter closely simulating the evaporative demand. however, a perfect correlation coefficient was not anticipated as the boundary - layer resistances between the wet wick and the plant crown are dissimilar, the thermal properties and heat capacity of the two systems differ, and the plant can control its diffusive resistance by changing stomatal aperture. on the other hand, a rippled, low - value cross - correlation function, with no clear peak, can be interpreted as a lack of dependence between the two time series, namely, the oscillations in wpt rate are autonomous and self - regulated, independent of the fluctuations in ambient conditions. typical variations in weight during the night and subsequent daylight hours are shown in fig. as water was supplied to the plants in the late evening (after 20.00 h), the weight decreased monotonically, albeit at different rates, during the night and daylight hours. the rate of the weight decrease (transpiration rate increase) intensified during the morning, stayed high during the noon and early afternoon hours, and weakened thereafter (fig . the wpt - rate time series ( the negative values of the rate of weight decrease), calculated by the time derivative of the measured weight - time series with data points every 3 min (equation 1), is shown in fig. a noisy wpt rate was obtained despite what appeared to be a relatively smooth pattern of weight decrease (fig . the amplitudes of the wpt rate were low during the night, early morning, and evening hours and large at the other times . a detailed pattern of the daily transpiration rate, especially when the ambient conditions varied with time, could barely be identified from this noisy time series . the smoothed wpt rate ( fig . 1b, solid line) was obtained by differentiating a smoothed weight - time series obtained by applying the s - g smoothing method with a window breadth of 30 data points (s - g 30). the fluctuations around the smoothed transpiration - rate pattern are usually considered random noise (white noise) associated with the measurement system, and as such are generally ignored without further analysis. here , however, these fluctuations were further analysed in order to isolate the system - noise - induced fluctuations from those induced by the ambient and/or physiologically driven oscillations. time - course of transpiration rate calculated by numerical derivative of the measured and smoothed time - courses of the decrease in potted tomato weight (b). the smoothed patterns of weight - variation rate (smoothed by s - g 30 prior to time differentiation) for a tomato plant, a wet wick, and a constant weight that was placed on a load cell are shown in fig. the whole - plant and wet - wick data were measured on 6 september 2006, and the constant load on 9 september 2006, all in the temperature - controlled greenhouse. 2a were measured for four neighbouring tomato plants that were grown together in the greenhouse (data not shown) with a similarly ample water supply. the residual time series (difference between the measured and smoothed weight time series) for the tomato plant, wet wick, and constant load are shown in fig. to exclude the possibility of the oscillations in wpt rate being simply system - related noise, these residuals were examined for randomness (white noise) by autocorrelation function (fig . the autocorrelation function of the constant - weight residual time series, k ( fig . 2i) was 1 at lag=0 and was close to zero for all other lags. by contrast, the autocorrelation functions of both the tomato plant and the wet wick (fig . 2 g, h, respectively) were periodic for the first 200 lags, with an approximate average 40 lag difference from peak to peak. the correlation between the fluctuations in plant weight around the smoothed weight variation and ambient conditions (represented by the wet wick) will be analysed further in the following. smoothed weight - variation - rate patterns in a whole potted tomato plant (a), a wet wick (b), and a constant load (c). residual time series for the whole plant (d), wet wick (e), and constant load (f). autocorrelation function of the residual time series for the whole plant (g), wet wick (h), and constant load (i). the spectra for the tomato - plant, wet - wick, and constant - load residual time series are shown in fig. 3e ) provided an additional indication that its residual time series is practically a random signal (white noise) while those of the plant and wet wick are not. 3a ) had high amplitudes at f < 2.5 h and lower amplitudes at the higher frequencies. the wet - wick spectrum (fig . 3c) exhibited a similar pattern, i.e. high amplitudes (although lower than those of the whole plant) at f < 2 h and lower amplitudes at the higher frequencies. to filter out what was considered to be white noise, both spectra were low - pass filtered for f < 2 h prior to further analysis. the time derivative of the reconstructed low - pass - filtered residual time series provided oscillations in wpt and wet - wick evaporation rates, as well as in the constant - load weight. superpositions of these oscillations on the smoothed variation of these variables are shown in fig. spectra for the whole - plant (a), wet - wick (b), and constant - load (c) residual time series. time derivative of the low - pass - filtered residual time series superimposed on the time derivative of the smoothed weight variation with time for the whole plant (d), wet wick (e), and constant load (f). to analyse the correlation between the fluctuations in wpt and wet - wick rates , the time derivatives of the reconstructed low - pass - filtered residual time series of these variables were plotted together in fig. 4a. the baseline in this figure (weight loss=0) was the smoothed transpiration / evaporation rate. a quantitative measure of this correlation was obtained by the cross - correlation function (fig . 4b), which is a standard method of estimating the degree to which two series are correlated. the cross - correlation between the two series was calculated for the morning (06.00 to 12.00 h) and afternoon (12.00 to 18.00 h) to examine the correlation between fluctuations in ambient conditions and the oscillations in wpt rate when transpiration rate increases (morning hours) and when it is already high and beginning to decrease (afternoon hours). when the cross - correlation around lag=0 is higher than those at other lag values (negative and positive), the oscillations in wpt rate can be more related to temporary variations (fluctuations) in ambient conditions. however, when low cross - correlation values are obtained at all lags, the two series are independent and the oscillations in wpt rate may be autonomous, putatively driven by independent physiological processes. the cross - correlation function for the morning hours had a slightly higher peak value than for the afternoon hours (fig . the presence of multiple peaks indicated that the oscillations in wpt rate depend on the fluctuations in ambient conditions, with periods when the oscillations in wpt rate are independent of those of the wet wick, namely, ambient conditions . 4a) revealed that they are temporarily out of phase between 07.30 and 10.00 h and between 13.30 and 15.30 h. from the smoothed transpiration pattern in fig. 3b, the shifts between the two oscillations coincided with periods of intensive change in transpiration rate be it increasing or decreasing. thus, it may be deduced that autonomous oscillations in wpt rate are associated with intensive variations in transpiration rate, even when water is fully available to the plant. oscillations in whole - plant transpiration (wpt) and wet - wick evaporation rates superimposed on the smoothed rates when the low - pass - filtered data are used to derive the transpiration and evaporation rates (a). cross - correlation function between transpiration and evaporation rates in the morning and afternoon hours (b). the effect of water - stress build - up on the momentary wpt rate (smoothed and oscillatory) is shown in fig. 5a and c is shown in fig. 5b and 5d, respectively, where the value of zero transpiration rate represents the smoothed transpiration rate in fig. 5a and 5c, the smoothed and superimposed oscillatory evaporation rates from the wet wick for the 5 d dehydration period are shown in fig. 5e and the isolated oscillations in evaporation rate (the difference between the smoothed and oscillatory patterns) for this period 5f. figure 5 indicates that (i) since these plants were grown in a temperature - controlled greenhouse with a repetitive pattern of daily ambient conditions (demonstrated by the repetitive daily pattern in wet - wick evaporation rate, fig 5e), water can be assumed to be fully available to the plant as long as the transpiration - rate pattern is similar to the previous day. accordingly, water seems to be fully available to plant i during the first 2 d of dehydration, and to plant ii only on the first day (fig . as opposed to plant i, whose transpiration rate decreased slightly on days 2 and 3 and substantially on days 4 and 5, the transpiration rate of plant ii decreased substantially on day 2 and was markedly low during the last 3 d of dehydration . differences in size between the two plants led to a significant difference in the maximum transpiration rate on day 1 ( 90 g h versus 160 g h for plants i and ii, respectively) and the amount of water remaining in the pots. (ii) similar to the transpiration - rate pattern in fig. 2a, the smoothed transpiration - rate patterns in fig. 5a and c have two daily peaks: a lower peak at noon followed by a higher one in the afternoon. note that the timing of the second daily peak coincides with the peak in daily evaporation rate. the two peaks in transpiration rate occurred despite the single daily peak in evaporation rate (fig . furthermore, the two peaks in transpiration rate were accompanied by a change in the pattern of transpiration - rate oscillations : the oscillations were amplified while the transpiration rate increased toward the first and even the second peak, and were shifted from their concurrence with the oscillations in evaporation rate ( figs 2a, 5a the increase in oscillation amplitude during this period contrasted with the uniform distribution of oscillation amplitudes in evaporation rate ( fig . ( iii) the ratio between the values of the two daily transpiration - rate peaks diminished with increasing water stress and was associated with the formation of a noticeable dip between the two peaks. (iv) the increase in oscillation amplitudes that was associated with transpiration - rate variation (increase in the morning hours or decrease in the afternoon hours) during the early stages of dehydration (fig . 5a, c) was also seen in the dip between the two daily transpiration - rate peaks as water stress progressed. smoothed and oscillatory whole - plant transpiration (wpt) rate for tomato plant i (a), isolated oscillations in wpt rate for tomato plant i (b), smoothed and oscillatory wpt rate for tomato plant ii (c), isolated oscillations in wpt rate for tomato plant ii (d), smoothed and superimposed oscillatory evaporation rates from the wet wick (e), and isolated oscillations in wet - wick evaporation rate (f), for five continuous days of dehydration. the oscillation amplitudes relative to the transpiration / evaporation rate (hereafter designated relative oscillations) are shown in fig. the relative oscillations are the ratio between the isolated oscillations in transpiration / evaporation rate and the smoothed transpiration / evaporation rate, respectively, in fig. 5. the relative oscillations in evaporation rate are larger in the early morning hours when the evaporation rate is low (fig . 5e), and then decrease as the evaporation rate increases and increase again in the late afternoon hours when the evaporation rate decreases again (fig . figure 6a d shows that the relative oscillations in transpiration increase during the dehydration period together with the decrease in transpiration rate ( fig . the oscillations in wpt rate may be temporary, and even decrease to zero ( relative transpiration= 1 in fig . 6b) under severe drought conditions when the transpiration rate is low. the daily pattern of relative oscillations in transpiration rate during the advanced dehydration stages (e.g. fig . 6b, d) indicates that they are relatively low in the morning, then intensify and stay high, even in the late afternoon hours. isolated oscillations in whole - plant transpiration (wpt) rate relative to the momentary values of the smoothed wpt rate for: plant i on day 1 (a) and day 4 (b) of dehydration, plant ii on day 1 (c) and day 4 (d) of dehydration. isolated oscillations in evaporation rate relative to the momentary values of the smoothed evaporation rate from the wet wick for day 1 (e) and day 4 (f) of dehydration. a comparison between the daily isolated oscillations in transpiration and wet - wick evaporation rates for dehydrating tomato plants i, together with the cross - correlation function for each dehydration day, are shown in fig. the cross - correlation function for each dehydration day for plant ii is shown (hollow circles) in fig. the cross - correlation function for plant i had an apparent single peak on day 1 (fig . this high cross - correlation value indicates that the synchronized oscillations in wpt rate were mostly induced by the fluctuations in ambient conditions in the greenhouse . however, there were no evident peaks in the cross - correlation function as water stress increased, which indicates that the synchronized oscillations in transpiration rate became self - regulated and independent of the fluctuations in ambient conditions demonstrated by the pattern of wet - wick evaporation . the decrease in transpiration rate along with the development of self - regulated oscillations in transpiration rate took place while the gap between water demand and water availability broadened . note that the self - regulated oscillations in plant i appeared on days 2 and 3 of dehydration ( fig . 7d, f), while the transpiration rates were similar to that on day 1 (fig . by contrast, the daily cross - correlation function for plant ii did not have any apparent single peak, not even on day 1 ( the hollow circles in fig . the differences in the cross - correlation functions between the two plants can be attributed to the high transpiration rate of plant ii on day 1 ( fig . plant ii was 6% heavier and had 7% higher leaf area than plant i. comparison between the isolated oscillations in tomato plant i transpiration and wet - wick evaporation rates and the respective cross - correlation function for these oscillations ( filled circles) during the 5 d dehydration experiment: day 1 (a and b, respectively), day 2 (c and d, respectively), day 3 (e and f, respectively), day 4 (g and h, respectively), day 5 (i and j, respectively). the cross - correlation function for plant ii during the 5 d dehydration experiment appear in b, d, f, h, and j, respectively as hollow circles. typical variations in weight during the night and subsequent daylight hours are shown in fig. as water was supplied to the plants in the late evening (after 20.00 h), the weight decreased monotonically, albeit at different rates, during the night and daylight hours. the rate of the weight decrease (transpiration rate increase) intensified during the morning, stayed high during the noon and early afternoon hours, and weakened thereafter (fig . the wpt - rate time series ( the negative values of the rate of weight decrease), calculated by the time derivative of the measured weight - time series with data points every 3 min (equation 1), is shown in fig. a noisy wpt rate was obtained despite what appeared to be a relatively smooth pattern of weight decrease (fig . the amplitudes of the wpt rate were low during the night, early morning, and evening hours and large at the other times . a detailed pattern of the daily transpiration rate, especially when the ambient conditions varied with time, could barely be identified from this noisy time series . the smoothed wpt rate ( fig . 1b, solid line) was obtained by differentiating a smoothed weight - time series obtained by applying the s - g smoothing method with a window breadth of 30 data points (s - g 30). the fluctuations around the smoothed transpiration - rate pattern are usually considered random noise (white noise) associated with the measurement system, and as such are generally ignored without further analysis. here , however, these fluctuations were further analysed in order to isolate the system - noise - induced fluctuations from those induced by the ambient and/or physiologically driven oscillations. time - course of transpiration rate calculated by numerical derivative of the measured and smoothed time - courses of the decrease in potted tomato weight (b). the smoothed patterns of weight - variation rate (smoothed by s - g 30 prior to time differentiation) for a tomato plant, a wet wick, and a constant weight that was placed on a load cell are shown in fig. the whole - plant and wet - wick data were measured on 6 september 2006, and the constant load on 9 september 2006, all in the temperature - controlled greenhouse. 2a were measured for four neighbouring tomato plants that were grown together in the greenhouse (data not shown) with a similarly ample water supply. the residual time series (difference between the measured and smoothed weight time series) for the tomato plant, wet wick, and constant load are shown in fig. to exclude the possibility of the oscillations in wpt rate being simply system - related noise, these residuals were examined for randomness (white noise) by autocorrelation function (fig . the autocorrelation function of the constant - weight residual time series, k ( fig . 2i) was 1 at lag=0 and was close to zero for all other lags. by contrast, the autocorrelation functions of both the tomato plant and the wet wick (fig . 2 g, h, respectively) were periodic for the first 200 lags, with an approximate average 40 lag difference from peak to peak. the correlation between the fluctuations in plant weight around the smoothed weight variation and ambient conditions (represented by the wet wick) will be analysed further in the following. smoothed weight - variation - rate patterns in a whole potted tomato plant (a), a wet wick (b), and a constant load (c). residual time series for the whole plant (d), wet wick (e), and constant load (f). autocorrelation function of the residual time series for the whole plant (g), wet wick (h), and constant load (i). the spectra for the tomato - plant, wet - wick, and constant - load residual time series are shown in fig. 3e ) provided an additional indication that its residual time series is practically a random signal (white noise) while those of the plant and wet wick are not. 3a ) had high amplitudes at f < 2.5 h and lower amplitudes at the higher frequencies. the wet - wick spectrum (fig . 3c) exhibited a similar pattern, i.e. high amplitudes (although lower than those of the whole plant) at f < 2 h and lower amplitudes at the higher frequencies. to filter out what was considered to be white noise, both spectra were low - pass filtered for f < 2 h prior to further analysis. the time derivative of the reconstructed low - pass - filtered residual time series provided oscillations in wpt and wet - wick evaporation rates, as well as in the constant - load weight. superpositions of these oscillations on the smoothed variation of these variables are shown in fig. spectra for the whole - plant (a), wet - wick (b), and constant - load (c) residual time series. time derivative of the low - pass - filtered residual time series superimposed on the time derivative of the smoothed weight variation with time for the whole plant (d), wet wick (e), and constant load (f). to analyse the correlation between the fluctuations in wpt and wet - wick rates , the time derivatives of the reconstructed low - pass - filtered residual time series of these variables were plotted together in fig. 4a. the baseline in this figure (weight loss=0) was the smoothed transpiration / evaporation rate. a quantitative measure of this correlation was obtained by the cross - correlation function (fig . 4b), which is a standard method of estimating the degree to which two series are correlated. the cross - correlation between the two series was calculated for the morning (06.00 to 12.00 h) and afternoon (12.00 to 18.00 h) to examine the correlation between fluctuations in ambient conditions and the oscillations in wpt rate when transpiration rate increases (morning hours) and when it is already high and beginning to decrease (afternoon hours). when the cross - correlation around lag=0 is higher than those at other lag values (negative and positive), the oscillations in wpt rate can be more related to temporary variations (fluctuations) in ambient conditions. however, when low cross - correlation values are obtained at all lags, the two series are independent and the oscillations in wpt rate may be autonomous, putatively driven by independent physiological processes. the cross - correlation function for the morning hours had a slightly higher peak value than for the afternoon hours (fig . the presence of multiple peaks indicated that the oscillations in wpt rate depend on the fluctuations in ambient conditions, with periods when the oscillations in wpt rate are independent of those of the wet wick, namely, ambient conditions . 4a) revealed that they are temporarily out of phase between 07.30 and 10.00 h and between 13.30 and 15.30 h. from the smoothed transpiration pattern in fig. 3b, the shifts between the two oscillations coincided with periods of intensive change in transpiration rate be it increasing or decreasing. thus, it may be deduced that autonomous oscillations in wpt rate are associated with intensive variations in transpiration rate, even when water is fully available to the plant. oscillations in whole - plant transpiration (wpt) and wet - wick evaporation rates superimposed on the smoothed rates when the low - pass - filtered data are used to derive the transpiration and evaporation rates (a). cross - correlation function between transpiration and evaporation rates in the morning and afternoon hours (b). the effect of water - stress build - up on the momentary wpt rate (smoothed and oscillatory) is shown in fig. 5a and c is shown in fig. 5b and 5d, respectively, where the value of zero transpiration rate represents the smoothed transpiration rate in fig. 5a and 5c, the smoothed and superimposed oscillatory evaporation rates from the wet wick for the 5 d dehydration period are shown in fig. 5e and the isolated oscillations in evaporation rate (the difference between the smoothed and oscillatory patterns) for this period are shown in fig. figure 5 indicates that (i) since these plants were grown in a temperature - controlled greenhouse with a repetitive pattern of daily ambient conditions (demonstrated by the repetitive daily pattern in wet - wick evaporation rate, fig 5e), water can be assumed to be fully available to the plant as long as the transpiration - rate pattern is similar to the previous day. accordingly, water seems to be fully available to plant i during the first 2 d of dehydration, and to plant ii only on the first day (fig . as opposed to plant i, whose transpiration rate decreased slightly on days 2 and 3 and substantially on days 4 and 5, the transpiration rate of plant ii decreased substantially on day 2 and was markedly low during the last 3 d of dehydration . differences in size between the two plants led to a significant difference in the maximum transpiration rate on day 1 ( 90 g h versus 160 g h for plants i and ii, respectively) and the amount of water remaining in the pots. (ii) similar to the transpiration - rate pattern in fig. 2a, the smoothed transpiration - rate patterns in fig. 5a and c have two daily peaks: a lower peak at noon followed by a higher one in the afternoon. note that the timing of the second daily peak coincides with the peak in daily evaporation rate. the two peaks in transpiration rate occurred despite the single daily peak in evaporation rate (fig . 5e). furthermore, the two peaks in transpiration rate were accompanied by a change in the pattern of transpiration - rate oscillations: the oscillations were amplified while the transpiration rate increased toward the first and even the second peak, and were shifted from their concurrence with the oscillations in evaporation rate (figs 2a, 5a the increase in oscillation amplitude during this period contrasted with the uniform distribution of oscillation amplitudes in evaporation rate ( fig . ( iii) the ratio between the values of the two daily transpiration - rate peaks diminished with increasing water stress and was associated with the formation of a noticeable dip between the two peaks. (iv) the increase in oscillation amplitudes that was associated with transpiration - rate variation (increase in the morning hours or decrease in the afternoon hours) during the early stages of dehydration (fig . 5a, c) was also seen in the dip between the two daily transpiration - rate peaks as water stress progressed. smoothed and oscillatory whole - plant transpiration (wpt) rate for tomato plant i (a), isolated oscillations in wpt rate for tomato plant i (b), smoothed and oscillatory wpt rate for tomato plant ii (c), isolated oscillations in wpt rate for tomato plant ii (d), smoothed and superimposed oscillatory evaporation rates from the wet wick (e), and isolated oscillations in wet - wick evaporation rate (f), for five continuous days of dehydration. the oscillation amplitudes relative to the transpiration / evaporation rate (hereafter designated relative oscillations) are shown in fig. the relative oscillations are the ratio between the isolated oscillations in transpiration / evaporation rate and the smoothed transpiration / evaporation rate, respectively, in fig. 5. the relative oscillations in evaporation rate are larger in the early morning hours when the evaporation rate is low (fig . 5e), and then decrease as the evaporation rate increases and increase again in the late afternoon hours when the evaporation rate decreases again (fig . figure 6a d shows that the relative oscillations in transpiration increase during the dehydration period together with the decrease in transpiration rate ( fig . the oscillations in wpt rate may be temporary, and even decrease to zero ( relative transpiration= 1 in fig . 6b) under severe drought conditions when the transpiration rate is low. the daily pattern of relative oscillations in transpiration rate during the advanced dehydration stages (e.g. fig . 6b, d) indicates that they are relatively low in the morning, then intensify and stay high, even in the late afternoon hours. isolated oscillations in whole - plant transpiration (wpt) rate relative to the momentary values of the smoothed wpt rate for: plant i on day 1 (a) and day 4 (b) of dehydration, plant ii on day 1 (c) and day 4 (d) of dehydration. isolated oscillations in evaporation rate relative to the momentary values of the smoothed evaporation rate from the wet wick for day 1 (e) and day 4 (f) of dehydration. a comparison between the daily isolated oscillations in transpiration and wet - wick evaporation rates for dehydrating tomato plants i, together with the cross - correlation function for each dehydration day, are shown in fig. the cross - correlation function for each dehydration day for plant ii is shown (hollow circles) in fig. the cross - correlation function for plant i had an apparent single peak on day 1 (fig . this high cross - correlation value indicates that the synchronized oscillations in wpt rate were mostly induced by the fluctuations in ambient conditions in the greenhouse . however, there were no evident peaks in the cross - correlation function as water stress increased, which indicates that the synchronized oscillations in transpiration rate became self - regulated and independent of the fluctuations in ambient conditions demonstrated by the pattern of wet - wick evaporation . the decrease in transpiration rate along with the development of self - regulated oscillations in transpiration rate took place while the gap between water demand and water availability broadened . note that the self - regulated oscillations in plant i appeared on days 2 and 3 of dehydration ( fig . 7d, f), while the transpiration rates were similar to that on day 1 (fig . by contrast, the daily cross - correlation function for plant ii did not have any apparent single peak, not even on day 1 ( the hollow circles in fig . the differences in the cross - correlation functions between the two plants can be attributed to the high transpiration rate of plant ii on day 1 ( fig . plant ii was 6% heavier and had 7% higher leaf area than plant i. comparison between the isolated oscillations in tomato plant i transpiration and wet - wick evaporation rates and the respective cross - correlation function for these oscillations ( filled circles) during the 5 d dehydration experiment: day 1 (a and b, respectively), day 2 (c and d, respectively), day 3 (e and f, respectively), day 4 (g and h, respectively), day 5 (i and j, respectively). the cross - correlation function for plant ii during the 5 d dehydration experiment appear in b, d, f, h, and j, respectively as hollow circles. the development of autonomous and self - regulated oscillations in wpt rate is postulated to be the plant response to the increasing gap between the evaporative demand and water availability. although the relative oscillations increased as water stress intensified (fig . d), their short - term variation was associated with the temporal variations in the between evaporative - demand to water - availability balance. the relative oscillations were low during the early morning hours, then increased with the increase in evaporative demand, and remained high during the afternoon hours when the evaporative demand had already decreased (fig . 6b, d). given that transpiration rate is highly dependent on temporal variation in soil water content (kramer and boyer, 1995 ; ray and sinclair, 1998 ; doussan et al . , 2006 ; garrigues et al ., 2006), the following scenario can be hypothesized to explain this irregular pattern of the relative oscillations. the moisture content around the plant root is usually lower at the end of the day and is gradually replenished during the night by mass flow from the wetter soil bulk. when the moisture content in the soil is high, this water flow from the bulk soil to the root vicinity also takes place during the daylight hours at a rate that enables the plant to catch up with the evaporative demand (first 2 d in fig . when the transpiration rate follows the evaporative demand, soil water is considered fully available at the given evaporative demand . however, when soil water becomes depleted, the flow rate from the soil bulk to the root vicinity decreases owing to an increase in the soil 's resistance to flow, and the transpiration rate decreases despite the high evaporative demand ( last 3 d in fig . when the soil becomes dry, the only available water that can cope with the evaporative demand is the amount around the roots in the morning hours when the replenished water there can handle the concurrent low evaporative demand . 6b, d). however, when the evaporative demand increases and water content in the root vicinity is nearly depleted with no replenishment from the soil bulk, the relative oscillation amplitudes intensify with decreasing transpiration rate (fig . plants are exposed to the risk of embolism under high evaporative demand, and particularly under drought stress owing to the increase in xylem tension, which is generally seen as a potentially catastrophic dysfunction of the axial water - conducting system ( tyree and sperry, 1989 ; tardieu and davies, 1993 ; hacke and sauter, 1995). one interpretation of the observations in this study is that the autonomous and self - regulated oscillations in wpt rate with higher relative amplitudes are designed to diminish or even prevent the increase in xylem tension. in which the relationship between acoustic emission, transpiration rate, and water stress in tomato plants was measured. reported that under mild or no conditions of water stress, the acoustic emission (representing the development of embolism) significantly increases with transpiration rate, both positively and negatively. this observation agrees with our finding that temporary autonomous oscillations in wpt rate develop when the transpiration rate is sharply increasing in the morning hours (and sharply decreasing in the afternoon) (figs 4a, 7a). showed that the acoustic emission increases with a decrease in the amount of soil water under mild or moderate conditions of water stress, and decreases with the decrease in the amount of soil water under severe water stress. other studies revealing a link between stomatal conductance, leaf water potential, and xylem embolism were performed by salleo et al. they suggested that xylem embolism may be a pre - signal for stomatal closure, thus representing an important step in the stomatal control of transpiration. the important role of the long (root - to - shoot) hydraulic signal in inducing stomatal closure under water stress has recently been reported (christmann et al ., 2007). the independent findings of a correlation between water stress and the formation of cavitation (salleo et al . , 2000 ; qiu et al ., 2002 ; and others) and the development of autonomous self - regulated oscillations in wpt rate under water stress suggest that these oscillations are related to the plant's response to increasing xylem tension. consequently, it is postulated that the oscillations in wpt rate are driven by a rapid, root - induced hydraulic signal that spreads instantaneously throughout the entire vascular system and inter alia, regulates stomatal aperture. could the oscillations in wpt rate be related to short - term growth of the plant? tomato plants maintain almost constant weight during the day, while their growth periods take place from the late afternoon to early morning (menzel et al . tomato plants present ishohdric behaviour ( sade et al ., 2009), namely, maintaining constant leaf relative water content during midday, under high available water conditions and drought condition alike. combining this intrinsic property with the observations that the autonomous and self - regulated oscillations in wpt appeared only under water stress, suggest that the measured oscillations were not induced by short - term oscillations in the plant's growth. in , synchronized autonomous self - regulated oscillations in wpt rate with periods of 2050 min were measured for tomato plants under water stress. these oscillations were interpreted as changes in leaf conductance triggered by hydraulic signals spread instantaneously via the plant vascular system which ed from drought - induced xylem tension. though the role of the oscillations is not yet clear, a plausible assumption is that they are a means used by the plant to control xylem tension while preventing the critical tension threshold that impels embolism.

plants respond to many environmental changes by rapidly adjusting their hydraulic conductivity and transpiration rate, thereby optimizing water - use efficiency and preventing damage due to low water potential. a multiple - load - cell apparatus , time - series analysis of the measured data, and residual low - pass filtering methods were used to monitor continuously and analyse transpiration of potted tomato plants (solanum lycopersicum cv . ailsa craig) grown in a temperature - controlled greenhouse during well - irrigated and drought periods. a time derivative of the filtered residual time series yielded oscillatory behaviour of the whole plant's transpiration (wpt) rate. a subsequent cross - correlation analysis between the wpt oscillatory pattern and wet - wick evaporation rates (vertical cotton fabric, 0.14 m2 partly submerged in water in a container placed on an adjacent load cell) revealed that autonomous oscillations in wpt rate develop under a continuous increase in water stress, whereas these oscillations correspond with the fluctuations in evaporation rate when water is fully available. the relative amplitude of these autonomous oscillations increased with water stress as transpiration rate decreased. these support the recent finding that an increase in xylem tension triggers hydraulic signals that spread instantaneously via the plant vascular system and control leaf conductance. the regulatory role of synchronized oscillations in wpt rate in eliminating critical xylem tension points and preventing embolism is discussed.

functional limitation of patients with heart disease has been documented consistently by cross - sectional and longitudinal studies. in a recent study conducted in 15 countries, with no exception, heart disease was associated with poor subjective health, above and beyond the effect of socioeconomics. the study, however, showed cross - country differences in the interactions between socioeconomic factors and heart disease in shaping well - being of populations. heart disease had a larger effect on subjective health of the elderly in the u.s. and china, women in the u.s., south africa, and india, low - income people in china and costa rica, and individuals with low education in uruguay and ghana. most of the research on the determinants of disability and the well - being of patients with heart disease has focused on either psychological, clinical, or behavioral characteristics. thus, less is known about the additive effects of social, behavioral, and comorbid conditions. multiple aspects of the daily life of patients with heart disease may be influenced by the condition. heart disease may be accompanied with a wide range of symptoms (e.g. dyspnea, tiredness, and fatigue) leading to functional limitation. patients with heart disease experience limitations in activities of daily living (adl). impaired functional capacity and. additional research on the effect of the social determinants of the well - being of patients with heart disease is needed. old age is associated with limitation in function and impaired well - being, physical, and mental health. gender also influences perceived health, with women tending to report higher levels of disability and morbidity. education and income, the most commonly accepted proxies of socioeconomic position, are associated with subjective health, chronic disease, and mortality. physical activity and exercise reduce the likelihood of health - related disability, especially during old age, and improve health - related quality of life. drinking, smoking, and physically inactive life style carry individual risks to adl, especially later in life. chronic medical conditions associated with heart disease are also major causes of morbidity and mortality. most studies have documented lower health and well - being, functional status, and health - related quality of life in the presence of chronic medical conditions. although we already know that patients with heart disease experience and report functional limitations, the contribution of various determinants on disability may differ across countries. unfortunately, our information is very limited about cross - country differences in the additive effects of determinants of disability associated with heart disease. in response to the gap of knowledge on cross - country variations in the determinants of disability among patients with heart disease, we compared countries for the additive effects of social, behavioral, and medical determinants and disability among older adults with heart disease. we used publicly available data of the research on early life and aging trends and effects (relate), a collection of multiple surveys from different countries across the world. the relate data are composed of the following surveys: 1 ) wisconsin longitudinal study; 2 ) china health and nutrition study; 3 ) chinese longitudinal healthy longevity survey (clhls); 4 ) costa rican study of longevity and healthy aging; 5 ) puerto rican elderly: health conditions; 6 ) study of aging survey on health and well being of elders; and 7 ) who study on global ageing and adult health. the sample size distribution of each country in the publicly available data is presented in table 1. sample size distribution of the participating countries in the relate data the original relate study enrolled more countries than were entered into the current analysis. this manuscript is limited to data from china, costa rica, puerto rico, mexico, barbados, brazil, chile, cuba, uruguay, india, ghana, south africa, and russia relate, research on early life and aging trends and effects (relate); creles, costa rican longevity and healthy aging study; prehco, puerto rican elderly: health conditions; sabe, survey on health, well - being, and aging in latin american and the caribbean; who, world health organization; sage, study on global ageing and adult health; chns, china health and nutrition survey; clhls, chinese longitudinal healthy longevity survey the participating countries represent a diverse range of national income levels and were selected from multiple continents. ghana represents low - income countries; china and india represent lower middle - income countries; cuba, uruguay, chile, costa rica, brazil, mexico, and russia represent upper middle - income countries; and puerto rico and barbados represent high - income countries. although the original relate study included a few other countries as well, countries participating in the current analysis were limited to those with available data on our variables of interest and included china, costa rica, puerto rico, mexico, barbados, brazil, chile, cuba, uruguay, india, ghana, south africa, and russia. the presence of self - reported physician diagnosis of heart disease and age over 65 years were considered as eligibility criteria. self - reported data on physician - diagnosis of chronic medical conditions such as heart disease is valid and closely associated with the physician - diagnosis of heart disease and medical record data. the socioeconomic data included age (continuous variable), gender (dichotomous variable), education (continuous variable), and income (continuous variable). income in this study was per capita annual household income calculated as purchase power parity dollars (ppp$). to provide the ppp$ or international dollar, costs (or incomes) in local currency units an international dollar is a hypothetical currency that is used as a means of translating and comparing costs from one country to the other using a common reference point, the us dollar. the ppp$ exchange rates are provided by the world health organization. a ppp$ exchange rate can be defined as the number of units of a country's currency required to buy the same amounts of goods and services in the domestic market as the u.s. the number of comorbid medical conditions was calculated based on the presence of self - reported physician diagnosis of diabetes, respiratory conditions, stroke, hypertension, and arthritis. self - reported data on chronic medical conditions are believed to be in agreement with physician diagnosis of conditions (kappa : 0.740.92). we approached physical disability from an operational point of view, focusing on limitations in adl. the adl items included in this study comprised bathing, getting dressed, going to the toilet, transferring, lifting heavy objects, shopping, and eating meals. informed consent was also provided by all the participants of all the surveys. for the statistical analyses, the statistical software spss version 20.0 for windows (spss inc ., chicago, il) was used. as weights were not applicable to surveys from china (chns), we did not apply sampling weights. socioeconomic factors (age, gender, education, and income), health behaviors (exercise, smoking, drinking), and number of chronic medical conditions (hypertension, respiratory, arthritis, stroke, and diabetes) were entered into country - specific hierarchical logistic regressions. in the first step (model i) , we tested the main effects of socioeconomic factors. in the next step (model ii), we also entered health behaviors. in the third step (model iii), we also included the number of chronic medical conditions. odds ratios (ors) and 95% confidence intervals (95% ci) were reported. the socioeconomic factors of the participants in each country have been reported elsewhere. in model i, high age was predictive of adl limitation in all the countries other than uruguay, ghana, and south africa. female gender was not associated with adl limitation in most countries, with the exception of mexico. in south africa, high income was linked to lower odds of adl limitation only in costa rica and puerto rico. in chile, higher education was associated with lower adl limitation in mexico, india, and russia. in chile, cross - country differences in associations between socioeconomic factors and disability among patients with heart disease as table 3 depicts, in model ii, only in 4 countries (i.e. china, puerto rico, brazil, and cuba) was exercise associated with lower adl limitation. in 3 countries (i.e. india, costa rica, and mexico), the association between exercise and adl limitation was marginally significant. with a few exceptions (i.e. china, brazil, chile, and uruguay), most countries did not show an association between drinking and adl limitation. smoking was not associated with adl limitation among individuals with heart disease, above and beyond the socioeconomic factors, exercise, and drinking. cross - country differences in associations between socioeconomic factors and health behaviors among patients with heart disease as table 4 demonstrates, in model iii, number of medical comorbidities was positively associated with odds of adl limitation in 10 countries. the number of medical comorbidities was marginally associated with adl limitation in one country (barbados). cross - country differences in associations between socioeconomic factors, health behaviors, and medical conditions among patients with heart disease this study revealed major cross - country differences in the additive effects of socioeconomic, behavioral, and medical characteristics on disability among patients with heart disease. the number of medical comorbidities and age were predictive of disability in most countries, while gender and income were linked to disability in very few countries. surprisingly, smoking was not associated with disability in any of the countries, while socioeconomic factors and other health behaviors (i.e. exercise and drinking) were constant. to summarize, the number of comorbid medical conditions, age, physical activity, drinking, education, income, and gender were associated with disability in 85%, 85%, 54%, 31%, 31%, 23%, and 15% of the countries. there are very few previous studies to compare our findings with. based on a recent study that compared 15 countries, age in the u.s. and china; gender in the u.s., south africa, and india; income in china and costa rica; and education in uruguay and ghana modified the effect of heart disease on subjective health. in puerto rico, argentina, barbados, brazil, chile, cuba, and russia, the effect of heart disease on subjective health was above and beyond the influence of socioeconomic factors. the findings of a recent in press study revealed that countries largely vary in the contributors of adl limitation in the general population. the study particularly found considerable cross - country differences for the relationship between age and adl. the contribution of age and gender in explaining the variance of adl was very high in china and cuba, respectively. more variation was seen in the effect of education than income as a factor contributing to the adl across countries. health behaviors such as exercise and also chronic conditions (in general) consistently explained a significant portion of the variance of adl across all the 8 countries included in that study. based on our study, age was linked to disability among individuals with heart disease in 10 of the 13 countries. age is known to be positively associated with adl limitation. in almost all countries, individuals with diabetes are more likely to experience restrictions in adl, along with reduced mobility and role functioning. a recent study documented a significant correlation between the comorbidity score and all the measures of well - being among patients with ischemic heart disease. the comorbidity score was correlated with physical and mental quality of life, psychological distress, sleep quality, and dyadic adjustment. authors emphasized that primary health care physicians, family physicians, and cardiologists have a major role in identifying and treating comorbid somatic conditions among patients with ischemic heart disease. according to a cross - country study, in all countries and with no exception, heart disease was associated with higher odds of poor subjective health, above and beyond the effect of age, gender, education, and income. this is in line with previous studies suggesting the role of heart disease on well - being, quality of life, and disability. in a study, well - being was mostly affected by heart conditions, followed by asthma / chronic bronchitis, joint complaints, back problems, and diabetes. another study suggested that heart diseases, musculoskeletal diseases, lung diseases, neurological disorders, diabetes, and cancer may have more influence on disability at the population level, compared to other conditions. another study showed that patients with heart disease, as well as patients with hearing impairment, neurological disease, and vision impairment, report the highest levels of distress. a study also showed that after controlling the effect of age, sex, educational level, comorbidities, disability and pain, coronary artery disease and chronic hemodialysis were linked to the highest levels of depression. according to a cross - country study, heart disease was the only factor consistently associated with poor perceived health among individuals with diabetes. only in two countries, women report lower levels of quality of life, whereas men have lower mortality. in general the current study also documented cross - country differences in the association between education and income and adl limitation among elderly with heart disease. based on the current study, clinicians in different countries may need to consider different socioeconomic and behavioral factors to estimate or reduce disability (adl limitation) among patients with heart disease. based on our findings, locally designed health promotions may be superior to universal programs for patients with heart disease. in almost all countries, however, disability may be reduced if comorbid medical conditions are properly diagnosed and treated. that is, attention to comorbid conditions may be considered as a common component of disability prevention for patients with heart disease. similar to other studies, the current study is limited in several ways. due to the cross - sectional design, health behaviors such as smoking, drinking, and exercise were measured using single items. only a few comorbid medical conditions were included, and the type of conditions was not entered into the model. to conclude, there are major cross - country differences in the determinants of disability among patients with heart disease. the findings advocate designing and implementing country - specific programs to reduce disability among patients with heart disease.

abstract: patients with heart disease experience limited activities of daily living (adl). this is a cross - country comparison of the additive effects of socioeconomics, health behaviors, and the number of medical comorbidities on disability among patients with heart disease.methods:the current study used a cross - sectional design. data came from the research on early life and aging trends and effects (relate). the current analysis utilized data on elderly individuals (age 60 y) from 13 countries. the outcome was any adl limitation (i.e. bathing, dressing, using toilet, transferring, lifting heavy things, shopping, and eating meals). socioeconomics (i.e. age, gender, education, and income), health behaviors (i.e. exercise, smoking, and drinking), and number of chronic medical conditions (i.e. hypertension, respiratory, arthritis, stroke, and diabetes) were entered into country - specific logistic regressions, considering at least one limitation in adl as the main outcome.:number of comorbid medical conditions and age were positively associated with disability in 85% of the countries. physical activity and drinking were linked to disability in 54%and 31% of countries, respectively. higher education and income were associated with lower disability in 31% and 23% of the countries, respectively. female gender was associated with higher disability only in 15% of the countries. smoking was not associated with disability, while the effects of socioeconomics, drinking, exercise, and medical comorbidities were controlled.:determinants of disability depend on the country; accordingly, locally designed health promotion interventions may be superior to the universal interventions for patients with heart disease. medical comorbidities, however, should be universally diagnosed and treated.

the effects of whole body irradiation (wbr) on particle clearance and the development of pulmonary fibrosis have been investigated. using carbon, clearance is accomplished by polymorphonuclear leukocytes (pmn) and alveolar macrophages (am), and only a few particles reach the interstitum. however, in preirradiated mice, the usual eflux of inflammatory cells is much delayed so that more free carbon remains in the alveoli, and by 1 week, many particles cross the epithelium to be phagocytized by interstitial macrophages. carbon is found in the peribronchiolar interstitium 6 months later with no evidence of fibrosis. in the present study, mice received 1 mg silica intratracheally 2 days after 6.5 gy wbr when the white blood cell count was low. a much - reduced am and pmn response was found in the following 2 weeks compared to the reaction to silica alone, and many silica particles reached interstitial macrophages. in this case, macrophage activation by silica was associated with fibroblast proliferation, and by 16 weeks, much more pulmonary fibrosis was produced than after silica or irradiation only. this was measured biochemically and correlated with a large increase in retained silica in the irradiation - silica group. the indicate that radiation inhibits the inflammatory response to particle instillation, ing in greater translocation of free particles to the pulmonary interstitium. in the case of silica, the greater, prolonged interaction with interstitial macrophages leads to a much exaggerated fibrotic reaction.imagesfigure 3.figure 4.figure 5.figure 6.figure 7.

in high - income countries, chronic vascular disease is the leading cause of limb loss. however, many previously healthy individuals undergo amputation secondary to trauma, neoplasia, infection, and arterial embolism. the conventional way to suspend a prosthetic limb to the body is with a prosthetic socket. users of socket prostheses commonly report impaired quality of life and complications including dermatitis and infected sores. our novel method is based on the principles of osseointegration (fig . 1), which has been in clinical use in prosthetic teeth replacement since 1965. one report suggests a 15-year implant survival rate of approximately 90% in mandibular bone. with osseointegration, direct contact between fixture and bone tissue is intended, assuring a stable, long - term attachment for the external prosthesis. this technique in easy prosthesis handling, improved limb control, and eliminates socket - impaired rom and socket - caused skin disorders (fig . 2). during the years, various attempts to anchor prosthetic limbs with transcutaneous metal implants other than titanium have failed, primarily owing to infection. currently, our center has treated more than 100 patients with femoral titanium implants (fig . 3), three with tibial implants, 15 with humeral implants, and 20 with ulnar and/or radial implants. treatment failures were more common before we introduced a standardized treatment protocol in 1999.fig. 2this is a ventral view of the transcutaneous component (abutment) in a patient with a transfemoral amputation. (published with permission from nigel jarvis, the university of gothenburg, the sahlgrenska academy, gothenburg, sweden.)fig. 3a lateral view is shown of a patient securing an external prosthesis to the abutment. (published with permission from nigel jarvis, the university of gothenburg, the sahlgrenska academy, gothenburg, sweden .) this schematic drawing shows the implant components and surrounding tissues. this is a ventral view of the transcutaneous component (abutment) in a patient with a transfemoral amputation. (published with permission from nigel jarvis, the university of gothenburg, the sahlgrenska academy, gothenburg, sweden .) a lateral view is shown of a patient securing an external prosthesis to the abutment. (published with permission from nigel jarvis, the university of gothenburg, the sahlgrenska academy, gothenburg, sweden .) clinical observations have led us to believe that poor primary osseointegration increases the risk of subsequent infection. however, experimental evidence supports antiinfectious properties of titanium compared with other biomaterials when implanted in bone and in soft tissues. medical device infection is caused predominantly by staphylococci, with coagulase - negative staphylococci being the most common. however, as the definitive diagnosis of infection is sometimes a problem, culture - based algorithms have been developed to increase diagnostic sensitivity. we therefore base our definitions of infection on clinical, radiographic, and bacteriologic findings. although current titanium transcutaneous implants are intended to reduce infections compared with previously used metals and transcutaneous techniques, there are little data regarding infections with these implants. in the related but incomparable setting of dental osseointegration , infection is well characterized, but less has been published regarding infection in bone - anchored hearing aids. our aims were to describe the frequency and the bacterial flora at the skin - penetration area and its relation to the development of local and implant - related infection in this novel method. we prospectively studied bacterial colonization and infectious complications in 39 patients with arm and leg amputations previously treated with 45 transcutaneous osseointegrated titanium implants. all patients attending the osseointegration outpatient clinic at sahlgrenska university hospital, gteborg, sweden, for scheduled or emergency visits between january and june 2005 were included. at least 3 months had elapsed since the second surgical procedure (abutment insertion). this cohort was followed longitudinally for an average of 3 years to identify implant infections and cross sectionally surveyed twice (at inclusion and after approximately 3 years) for bacterial presence, local infection, and antibiotic use. all skin - penetrating loci were subjected to separate clinical, bacteriologic, and radiographic assessment. there were 18 women and 21 men with a mean age of 49.3 years (range, 2874 years). thirty - three of the implants were femoral (bilateral in one), four each were ulnar and radial (bilateral in one), three were humeral, and one was tibial. the patients had been living with the implant(s) a mean of 54 months (range, 3132 months). indications for these implants were severe discomfort when using conventional socket prostheses or poor stump conditions. is inserted into the residual bone and allowed to integrate for 6 months before the skin - penetrating extension (abutment) is inserted. based on the experience of successful skin - penetrating implants in the head and neck regions, the skin is attached directly to the distal end of the residual bone to reduce soft tissue mobility and risk of infection. we consider the implant osseointegrated when it is stable on clinical examination, pain free when loaded, and there are no signs of loosening seen on radiographs, ie, no radiolucent zone around the implant. postoperative followup includes clinical examination (pain evaluation, implant stability, skin and soft tissue condition), a rehabilitation protocol, and radiographs at 6 months and 1, 2, 3, 5, 7, 10, and 15 years after surgery. we used the following five definitions for implant infection and required all stated criteria for the given diagnosis: definite implant infection: (a) clinical symptoms of implant - related infection, (b) radiographic signs consistent with periimplant bone infection, and (c) at least three of five intraoperatively obtained cultures yielding identical pathogens; probable implant infection: (a) clinical symptoms of implant - related infection, (b) radiographic signs consistent with periimplant bone infection, and (c) positive relevant culture not as defined previously; possible implant infection: (a) clinical symptoms of implant - related infection, (b) radiographic signs consistent with periimplant bone infection, and (c) no relevant cultures; local infection in the skin penetration area: (a) local signs / symptoms of infection (inflammation with or without secretion) in the skin penetration area but no symptoms of deep infection, (b) no radiographic signs consistent with periimplant bone infection, and (c) with or without relevant cultures; bacterial colonization around the skin - implant interface: (a) neither local inflammatory signs nor other clinical symptoms of infection, with or without secretion, at the skin - implant interface, (b) no radiographic signs consistent with periimplant bone infection, and (c) positive bacterial culture from the skin - implant interface. we considered radiographic evidence of osteolysis with or without periosteal sclerosis around a previously integrated implant to be consistent with implant infection. the definitions also were based on previously proposed culture diagnostics in infected hip and knee arthroplasties. at inclusion patients were examined clinically and asked to answer a questionnaire (appendix 1) regarding infectious complications and antibiotic use during the 6 months preceding the visit. without any preparation of the surrounding skin , samples were taken from the skin - implant interface with a sterile cotton swab, transported in a coal - based medium, and cultured on routine agar plates for at least 2 days. a second set of bacterial cultures patients who were not scheduled for a visit at this time collected bacterial samples (cotton swabs) themselves according to careful written instructions and, if necessary, with the assistance of their attending clinic. these patients were experienced in handling the skin - implant area and we anticipated no methodologic problems with the patients performing their own cultures. cultures and questionnaires were sent to sahlgrenska university hospital by mail. sampled cultures transported in a coal - based medium the number of colony forming units (cfu) was defined as + + + (> 100 cfu), + + (10100 cfu), or + (< 10 cfu). a routine disk method was used to determine antibiotic resistance. the latest scheduled or symptom - prompted radiographs at inclusion and followup were checked for signs of bone infection. two already had osteomyelitic changes at the beginning of the study, four were excluded, and two were without infectious symptoms. the implant was extracted owing to mechanical loosening in a previously radiated femur (october 2006). in another patient, two patients (one swedish) did not complete the followup protocol for nonmedical reasons. the frequencies for patients with definite / probable / possible implant infections were 5% (two of 39) at inclusion and 18% (seven of 39) at followup (table 1). seven patients had a history of local infection at the skin penetration area during the 6-month period before inclusion and 11 patients had local infections during the 6-month period before followup (table 1). four and six patients, respectively, had been treated with short - term oral antibiotics. the secretion was purulent.table 1bacterial colonization and infection at the beginning of the study and at followuptype of infectioninitial assessmentfollowup (2 - 3 years)possible / probable / definite implant infection26local soft tissue infection in the skin penetration area*711superficial colonization without signs of infection23 16no growth of bacteria or signs of infection72lost for followup4 * including all episodes of infection during 6 months before first and second assessments; figures in parentheses correspond to total number of clinical sites, which exceed the number of patients; three patients had more than one implant; total number of implant infections during the 3-year study was seven; one patient had been treated successfully at followup; contact with admitting clinic indicated no implant infection. bacterial colonization and infection at the beginning of the study and at followup * including all episodes of infection during 6 months before first and second assessments; figures in parentheses correspond to total number of clinical sites, which exceed the number of patients; three patients had more than one implant; total number of implant infections during the 3-year study was seven; one patient had been treated successfully at followup; contact with admitting clinic indicated no implant infection. the most common bacteria found around the skin - implant interface were staphylococcus aureus, coagulase - negative staphylococci, and streptococci group a, b, or g. in three of seven patients with implant infections at followup, inclusion cultures from the skin - implant interface yielded the same species as the suspected infectious agent (table 2). staphylococcus aureus was cultured from specimens from 16 patients (17 implants), coagulase - negative staphylococci from 10, streptococcus group b, g, or nontypable from nine, beta - hemolytic streptococcus group a from one, enterococcus sp. from three, alfa streptococci from one, and gram - negative rods from four (table 2) at inclusion. no staphylococcus aureus strain was methicillin-resistant.table 2bacterial findings at the skin - implant interface at first observation and second observations 2.5 to 3 years later*bacteriafirst observation (n = 39)second observation (n = 30)not quantifiednumber of patients+++bacterial coloniesnumber of patients+++bacterial colonies++++++s. streptococci101011coryneforms000011000no growth80excluded / no culture9 * several patients had more than one bacterial species isolated; one patient had a positive s. aureus culture from two loci; cons = coagulase - negative staphylococci; several patients had growth of two or more bacteria at the same site. bacterial findings at the skin - implant interface at first observation and second observations 2.5 to 3 years later * * several patients had more than one bacterial species isolated; one patient had a positive s. aureus culture from two loci; cons = coagulase - negative staphylococci; several patients had growth of two or more bacteria at the same site. followup culture specimens were obtained from 30 patients, as five patients did not send specimens as instructed. staphylococcus aureus was still the most common finding followed by coagulase - negative staphylococci, streptococcus group b, serratia sp. enterococcus sp., alfa streptococcus, coryneforms, and pseudomonas aeruginosa (one each) (table 2). eight of 13 patients with recultured specimens with staphylococcus aureus at inclusion also had staphylococcus aureus isolated at followup. the clinical presentation varied. two patients had chronic skin fistulas to the implant with occasional secretion but without pain, fever, or implant loosening. fistulas were present more than 5 years before inclusion and were not treated with antibiotics during the observation time. it was verified by biopsies of the culture specimens and treated with ciprofloxacin for 6 months. distal infection involving bone and soft tissue was seen in two patients with good primary osseointegration. the etiology was mixed with staphylococcus aureus / coagulase - negative staphylococci and staphylococcus aureus / enterococcus faecalis being suspected. acute osteomyelitis occurred in one patient who had no fever but had acute pain develop in an osseointegrated femur. intraoperative cultures yielded staphylococcus aureus and coagulase - negative staphylococci (table 3). in two of seven patients with implant infections at followup, prosthetic use was not affected at any time, three patients were affected only briefly during the time around surgical intervention, and for the patient with acute osteomyelitis, the treatment outcome is still pending.table 3etiology and outcome of patients with implant infections at followupage of patient (years)months since surgical session 2 at inclusionmonths to infection after surgical session 2locussuspected bacterial etiology*culture at skin penetration areatreatment given at followupoutcome36335femurs. aureusno treatmentongoing, fistulas * cultures taken from tissue specimens or from fistulas; bacterial swab taken at initial assessment from the area where the implant system penetrates the skin; cons = coagulase - negative staphylococci; gbs = group b streptococci. etiology and outcome of patients with implant infections at followup * cultures taken from tissue specimens or from fistulas; bacterial swab taken at initial assessment from the area where the implant system penetrates the skin; cons = coagulase - negative staphylococci; gbs = group b streptococci. transcutaneous osseointegrated titanium implants for prosthetic systems in patients with amputations provide improved performance and less socket - caused complications in selected patients. our aim was to describe the frequency, clinical presentation, and bacterial occurrence in local and implant - related infections. first is the relatively short followup, although some patients had been living with the implant for more than 10 years. most patients were young or middle - aged and probably will require use of their implants or other prostheses for numerous decades. rather, we studied a subgroup of 39 patients recruited during a 6-month period. as all patients attending (regular and emergency visits) the clinic during the inclusion period agreed to participate, we presume this cohort represents the entire population. a retrospective analysis of implant infection and risk factors in 100 patients treated with femoral osseointegrated implants and a prospective analysis of patients treated with the refined protocol are in progress. skin - penetrating implants were long regarded as unattainable owing to the high rates of failure caused by infection. seven of 39 (18%) patients either had an implant infection or one developed within 3 years. the implant - infection / implant - year ratio (7/135) is slightly less (5%) than comparable from queen mary s hospital in london where four of 16 patients treated with the same method had an implant - related infection develop during a cumulative 67-year period (6%) (unpublished data, sooriakumaran s, robinson kp, ward da, darcy r, chittoor sn, written communication from 12th ispo congress in vancouver, canada, july 29, 2007). in our cohort, only one of seven patients was diagnosed and treated for an implant - related infection earlier than 31 months (mean, 34 months) after the surgical procedure. with arthroplasty, late infection (> 24 months) is considered more indicative of hematogenous seeding compared with early (< 3 months) or delayed (324 months) infections. in our four cases with more virulent bacteria, late onset of symptoms clearly indicates that intraoperative contamination was not the cause of infection. however, we can not know whether some of the infections were either of hematogenous or new origin, rather than as a of residual surgical contamination. we assessed the presence of local skin infections twice, with a greater frequency at followup (table 2). these adverse effects must be compared with those of conventional socket prostheses. for patients using conventional prostheses, socket - related problems such as ulcers and rashes on the residual limb and restricted movement dermatologic problems were reported to occur among 34% to 41% of those using socket prostheses. , no socket is needed to suspend the artificial limb and consequently no patient in our study had ulcers or contact dermatitis on the residual limb. although various microorganisms may cause foreign body - related infection, staphylococci are the most frequently isolated pathogens. in our study, approximately of the patients were colonized with potentially virulent staphylococcus aureus in the skin penetration area. despite this, only three patients had a staphylococcus aureus implant infection. the ability to adhere to the implant material and to promote biofilm formation are important pathogenic properties for staphylococci and other bacteria. the biocompatibility is greater with titanium compared with stainless steel and cobalt - chrome alloys. in osseointegration, the tight junction formed between the titanium and the bone tissue might prevent adhesion, colonization, and subsequent biofilm formation. osseointegration also can be established and maintained in aggressive inflammatory environments such as experimental arthritis in a rabbit knee model and in patients with rheumatoid arthritis. the osseointegration might explain why some patients with implant infections have such slow, or even no, progressive destruction of the bone tissue with subsequent loosening. it also may explain why patients with poor primary osseointegration are more susceptible to infectious complications. we found the osseointegration method with titanium implants and skin penetration of the titanium system in patients with leg and arm amputations caused few severe infectious complications. however, infectious complications occur in approximately two - fifths of the patients during a 3-year period, mostly as local infections in the skin penetration area and more rarely as low - activity implant - associated infections. staphylococcus aureus and coagulase - negative staphylococci were the most commonly cultured bacteria in the skin penetration area. in our opinion, the frequency and severity of infections do not appear to be obstacles for use of this method. however, future studies with longer followup will address risk factors and specific infectious complications in an attempt to reduce morbidity and increase usefulness of this novel treatment.

the concept of osseointegration involves direct contact between titanium implant and bone. this transcutaneous prosthetic system for amputees is intended to assure stable long - term fixation. most metal transcutaneous implants have failed, primarily owing to infection.questions/purposeswe determined the frequency and describe the presentation of infectious complications with this novel method. we also evaluated the bacterial flora at the skin - penetration area and its relation to the development of local and implant - related infection.patients and methodswe prospectively followed 39 patients with arm and leg amputations fitted with transcutaneous osseointegrated titanium implants a mean of 56 months earlier (range, 132133 months). there were 33 femoral, one tibial, four ulnar, four radial, and three humeral implants. patients were selected during a 6-month period in 2005 and identically reevaluated after 3 years. implant infection was defined as definite, probable, or possible based on clinical, radiologic, and microbiologic evidence.the frequency of implant infection was 5% at inclusion and 18% at followup. one patient with infection recovered owing to antibiotic treatment and another patient had the implant removed. most implant infections had low infectious activity, and in five of the seven patients with infections, prosthetic use was not affected. the most common bacteria in superficial and deep cultures were staphylococcus aureus and coagulase - negative staphylococci.despite frequent colonization around the skin - implant interface by potentially virulent bacteria such as staphylococcus aureus and bacteria associated with biomedical device infections such as coagulase - negative staphylococci, this titanium implant system for bone - anchored prostheses caused few infections leading to disability or implant removal.level of evidencelevel iv, therapeutic study. see the guidelines for authors for a complete description of levels of evidence.

in line with the worldwide aging trend, korea became an aging society in 2000 and is expected to become an aged society with an elderly population over the age of 65 of 14% of total population1. in addition, the average life span of koreans was 79.6 years in 2010, 15 years longer than that of 19801. despite increases in social demands by the elderly for high - quality and satisfactory life, with the increase in the elderly population and their extended average life span , there is a high possibility that the elderly will lives accompanied by more diseases because of the degree of prolongation of their life span2. especially, cerebrovascular diseases are known as typical geriatric diseases that threaten the health of the elderly. according to a survey by the korea center for disease control and prevention, 7.2% of the elderly population over the age of 65 had experience of stroke in 2013, which is an 5.3% increase from 20013. stroke is a generic term for both cerebral infarction caused by the blockage of a blood vessel in the brain, and cerebral hemorrhage caused by the rupture of a blood vessel in the brain. stroke is not only the most common of disease, but it also has enormous social costs, such as medical costs. medicine costs, loss of productivity, and the socio - economic cost of stroke in korea were estimated to amount to u$ 3.5 billion as of 20104. although stroke occurs in all age groups, the elderly are the most susceptible group, since its prevalence rate increases rapidly with every 10 years of age5. twenty - five percent of the stroke patients die within 1 month of stroke onset, and even after survival, it has been reported that 60% of the surviving patients suffer from various disabilities such as paralysis, motor disorder, cognitive impairment and speech disorders, which leave them unable to lead independent daily lives without the help of others. moreover, 70% of survivors suffer from communication disorders such as dysarthria and aphasia6, and among the disabilities ing from stroke, communication problems frequently occur. according to the american heart association and american stroke association, among 2 million stroke survivors in 2005, approximately 1 million had aphasia7. disabilities after stroke are known to have adverse effects on the quality of life8, and among them, communication disorders place burdens not only on patients but on their families as well9. as the majority of stroke patients have to lead their lives with disabilities, the quality of life including psychological and social aspects should be given consideration. especially, communication activities of daily living (c - adl) is important, because it examines the basic communication ability necessary for survival in daily living such as social interactions and non - verbal communication10. similarly, although communication is an important factor in terms of quality of life, studies on communication of stroke patients remain limited to the understanding of the linguistic abilities of stroke patients, and there is a lack of studies on the theme of communication. while studies of stroke patients activities of daily living (adl) have been regularly conducted, studies of c - adl are very rare. this study investigated the relationship between c - adl and the quality of elderly stroke patients lives to provide basic data for use in enhancing the quality of life. the present study conducted convenience sampling 4 times from august through december 2014, and surveyed 165 elderly over the age of 60, who were diagnosed as having stroke and receiving treatment in rehabilitation departments of general hospitals located in seoul, inchon, daejeon and gwangju. one hundred fifty - eight fully completed questionnaires were anaylzed; 7 questionnaires had incomplete answers. as the required minimum number of samples was 88 patients to obtain a significance level of =0.05, and an effect size of 0.5, with a statistical power of (1-)=0.95 according to the g - power 3.0 program, the number of subjects in this study was more than sufficient. the subjects were given sufficient explanation regarding the purpose and experimental method of this study before participation and gave their voluntary consent. the study protocol was approved by the institutional review board of n university and was conducted in accordance with the ethical principles of the declaration of helsinki. together with the questionnaire on communication activities of daily living, a survey of quality of life was conducted via individual interviews. the inclusion criteria for subjects were: literate patients with consciousness, within 1 year after the onset of stroke, who had no accompanying neurological diseases such as dementia or parkinson s disease, or apraxia or agnosia. stroke patients basic communication ability to survive in daily living was measured with the communication activities of daily living - second edition (c - adl-2)11. c - adl-2 is composed of a total of 50 questions with responses scored on a 3-point scale (0 point : wrong answer, 1 point : proper answer, 2 points : correct answer) and higher scores indicate higher communication ability in daily living. test questions are classified into following 7 categories: reading, writing or using numbers, social interactions, divergent communication, contextual communication, nonverbal communication, sequential relationships, and humor, metaphor, absurdity. among them , divergent communication includes deciding lunch time and choosing menus, and contextual communication contains interpretation of danger signs while sequential relationships includes understanding of speed limits and buying things in a supermarket. stroke patients quality of life was assessed using the stroke - specific quality of life (ssqol)12 developed by williams et al. ssqol is an index of stroke patients characteristics and comprises 49 questions in 12 areas including movement abilities (6 questions), functions of upper extremity (5 questions), linguistic function (5 questions), thinking abilities (3 questions), visual functions (3 questions), self - care (5 questions), emotional state (5 questions), personality (3 questions), physical stamina (3 questions), role in the family (3 questions), social role (5 questions) and productive capability (3 questions). each response is scored on a 5-point scale and higher scores indicate higher levels of quality of life. covariate variables were age, sex, final education (middle school and lower, high school or above), the average monthly income of households (less than 2 million won, 24 million won, more than 4 million won), marital status (living with spouse, living without spouse, unmarried person), drinking (non - drinker, former drinker, current drinker), smoking (non - smoker, past smoker, current smoker), stroke type (cerebral hemorrhage, cerebral infarction), time since onset of stroke (less than 3 months, 36 months, 612 months), depressive symptoms in the last 1 months (yes, no). first, for subjects general characteristics, means and percentages were calculated and are presented as descriptive statistics. second, in order to examine the differences in ssqol based on subjects c - adl characteristics, the independent t - test and one - way anova were performed. finally, the relationship between c - adl and ssqol was analyzed using multiple regression analysis. the average age of the subjects was 67.5 (standard deviation 6.8 years); 53.8% of the subjects were males, 88.5% were married, 63.6% were middle school graduates or above, 67.0% had cerebral infarction, 61.9% had a monthly household income of less than 2 million won, 26.5% were, past smokers, 38.5% were past drinkers, 68.0% had experience of depression symptoms in the last 1 month, and the majority of the subjects were 612 months since the onset of stroke. subjects mean c - adl was 62.3 points (standard deviation 16.5) and mean ssqol was 149.9 points (standard deviation 35.7). in the multiple regression analysis, the r of the final model was 0.23 and the explanatory power of the model was 23% with the variance inflation factors (vif) being all less than 10, proving that there was no problem with multi - collinearity among the independent variables. in the bivariate analysis, a significant positive relationship was found for c - adl with ssqol (b=0.54, t=13.28, p<0.05). in model 1, after adjusting for age, sex, final education, the average monthly income of households, marital status, drinking and smoking, the relationship of c - adl with ssqol was maintained (b=0.48, t=3.32, p<0.05). in model 2, after adjusting for all the compounding variables, c - adl had a significant positive relationship with ssqol (b=0.46, t=3.11, p<0.05). although communication problems frequently occur due to the after - effects of stroke, there is a lack of studies of stroke patients communication abilities in daily life. the present study investigated the relationship between elderly stroke patients c - adl and quality of life, and found that c - adl had a significant positive relationship with quality of life. numerous studies have reported a relationship between adl and quality of life. according to these studies, stroke patients adl is a significant variable predicting stroke patients quality of life13, and the higher the functions of adl, the higher the quality of life14. these are similar to the of the present study that higher stroke patients adl indicates higher quality of life. as disabilities from stroke become chronic, inability to independently perform adl, such as putting on clothes and eating for an extended period of time, not only causes helplessness and depression in stroke patients14, but also inflicts emotional pain, such as intellectual regression, despair and anxiety10. that is, functional disorders in daily life cause stroke patients to experience psychological pain and loss of social functions, and if these problems are prolonged, they are likely to cause deterioration in their quality of life and maladjustment in social relationships, changes in role and economic difficulties. although it is difficult to directly compare the of preceding studies10, 13, 14 with those of the present study, communication is likely to have a significant effect on quality of life, considering that it is an ability necessary for instrumental daily life. especially, stroke patients experience deterioration of social functions due to limitation of communication, and when they have difficulty in understanding the meaning of what another party says, or in producing speech, even when they have clear consciousness, it is highly possible that they feel extreme frustration and depression15. nevertheless, therapeutic rehabilitation for stroke mainly focuses on physical rehabilitation, while linguistic, emotional and social rehabilitation tends to be ignored. as communication problems are likely to lower the quality of life by leading stroke patients to give up their will to live16, in order to enhance stroke patients quality of life, it is necessary to consistently improve communication functions from the early stage after onset of stroke, and to extend support to reduce stroke patients difficulties in leading their daily lives. a limitation of this study was the exclusion of patients with severe aphasia, for whom test could not be conducted, therefore, caution should be taken in generalizing the of this study. also, since this study was a cross - sectional study, the can not be interpreted in causal relationships. a significant positive relationship with ssqol was found for c - adl in this study. this implies that it is necessary to enhance stroke patients communication ability in daily living in order to raise their quality of life.

disabilities after stroke are known to have adverse effects on the quality of life. this study investigated the relationship between communication activities of daily living (c - adl) and quality life of elderly stroke patients to provide basic data for use in enhancing the quality of life. one hundred sixty five elderly over the age of 60, who were diagnosed as having stroke and receiving treatment in rehabilitation departments of general hospitals were surveyed. stroke patients basic communication ability to survive in daily living was measured using the c - adl second edition, and stroke patients quality of life was measured with the stroke - specific quality of life (ssqol). the relationship between c - adl and ssqol was analyzed using multiple regression analysis. c - adl had a significant positive relationship with ssqol. this implies that it is necessary to enhance stroke patients communication ability in daily living in order to raise their quality of life.

the evolving scientific and regulatory activities in europe and north america emphasize the need for the development of tools that refine, replace, or reduce the use of animals and human volunteers in pharmacokinetic and toxicity tests. the ability to base the toxic responses on the target tissue dose or internal concentration of the toxic moiety of the chemicals is key to the predictive tools reflective of the current state of science. therefore, physiologically based pharmacokinetic (pbpk) models that are capable of providing a priori prediction of the time course of chemicals in blood and tissues is of tremendous interest. pbpk models are mechanistically based mathematical descriptions of the absorption, distribution, metabolism, and excretion of chemicals or pharmaceutical compounds. in pbpk models, the organism is represented as a set of several tissue compartments interconnected by blood flows. in these models, the internal dose measures (e.g., blood or tissue concentrations, amount metabolized) of a chemical are described on the basis of mass - balance differential equations requiring species - specific properties (e.g., alveolar ventilation rate, cardiac output, regional blood flows, and tissue volumes) and chemical - specific input parameters (e.g., partition coefficients and metabolic constants). although the species - specific values of several physiological parameters are available in the literature , the partition coefficients (pcs) and metabolic constants need to be determined experimentally or calculated by using animal - replacement methods for each chemical individually. the values of tissue: blood or tissue: plasma partition coefficients essential for developing pbpk models have been estimated for a wide range of chemicals and chemical classes, including drugs, with the use of tissue composition - based algorithms or qsar methods (e.g.,). regarding the metabolism parameters (i.e., hepatic clearance, intrinsic clearance, vmax, km, kcat, free energy of binding, energy of activation, or activation enthalpy), some studies have developed 2-d and 3-d qsars but with a specific focus on either a single isozyme, a single reaction or a single class of substances. none of these past efforts succeeded in predicting both vmax and km (or clint) of environmental chemicals for direct incorporation within animal or human pbpk models. alternatively, few studies utilized the group contribution method of gao , to predict metabolic rates for pbpk models. in this method, the chemical is decomposed into different structural fragments or groups, the contributions of which are obtained by regression analysis. accordingly, these publications demonstrated the feasibility of developing structure - property relationships for the metabolism rates. the group contribution method was successfully used to develop quantitative structure - property relationships (qsprs) for the tissue: air partition coefficients as well as intrinsic (clint) and hepatic clearance (clh for a group of low - molecular - weight volatile organic chemicals ( vocs) in rats. these qspr models, in turn, were incorporated within pbpk models to predict reasonably well the blood kinetics of inhaled vocs in rats. as these qsprs are species specific, they could not be used to conduct interspecies extrapolations. to overcome this limitation, bliveau et al. developed biologically based algorithms for pcs and clh to conduct rat to human extrapolations of the inhalation toxicokinetics of vocs. in this study, qsprs based on the group contribution method were developed for the chemical - specific input parameters of the biological algorithms for pcs (i.e., oil : air, water : air, and blood protein : air) and clint (intrinsic clearance normalized for cytochrome p450 2e1 content). more recently, qsprs were developed for the metabolic constants vmax (maximum velocity of reaction) and km (michaelis constant) and were further incorporated within a rat pbpk model to predict the toxicokinetics of mixtures of vocs. despite the successful use of the group contribution method in qspr modeling of metabolism rates, their principal limitation relates to the fact that the chemical space they cover is extremely limited (low - molecular - weight vocs containing one or more of the following fragments : ch3, ch2, ch, c, c = c, h, br, cl, f, benzene ring, and h on benzene ring). more experimental data on diverse chemicals would be needed to determine the contributions of other molecular fragments, as has been done with pow (e.g., estimation of the contribution of 130 fragments ( i.e., groups) required 1200 measurements of pow ). to extend the currently available qspr for clint to cover more diverse fragments and at the same time respect a reasonable ratio of the number of parameters to the number of observations, since the critical limitation in the construction of pbpk models for new substances continues to be the metabolism rate, a pragmatic approach particularly for inhaled vocs is to evaluate the maximum and minimum possible blood concentration profiles in exposed individuals. thus, using a hepatic extraction ratio (e) of 0 and 1 in the pbpk models, poulin and krishnan obtained simulations of the physiological limits (i.e., maximal and minimal blood concentration profiles) for inhaled vocs in humans. assuming the conceptual pbpk model and the values of its physiological parameters are reliable, the real answer , that is, the actual concentrations and kinetic curve, would be somewhere in between the theoretical limits simulated with these pbpk models. the uncertainty associated with these theoretical bounds can be reduced by developing better estimates of the metabolism constants. this could be done, at a practical level, by developing in silico tools that provide a range of plausible values, in lieu of a single accurate point estimate. such a tool might be of use for the toxicokinetic screening of substances, until the time when the chemical - specific measurements are obtained in vivo, in vitro, or with a highly precise mechanistic in silico method. since human exposures to environmental contaminants in most cases do not attain levels that approach or exceed saturation, it is not crucial to predict vmax and km separately, particularly for simulating kinetics in humans exposed to low atmospheric concentrations of vocs. therefore, the availability of in silico approaches based on easily available parameters to predict plausible range of clint would be desirable as a screening - level tool. the objective of this study was therefore to develop a quantitative property - property relationship (qppr) model of animal data to generate initial estimates (or bounds) of intrinsic clearance of vocs, for eventual incorporation within a human pbpk model to simulate blood concentration profiles associated with inhalation exposures. in this regard , we focused on evaluating the impact of the uncertainty associated with qppr predictions of clint on the blood kinetics of vocs in humans, relative to that of the uncertainty associated with the total lack of knowledge of the metabolic rate in humans. furthermore, the reliability of applying the qppr to predict the area under the blood concentration versus time curve (auc) of parent chemicals was evaluated, as a function of the sensitivity of the metabolism parameter in the pbpk model and the prediction uncertainty of qppr model. the qppr predictions were then compared with experimental data for several vocs and the pharmacokinetics in humans were simulated using integrated qppr - pbpk models for these 26 vocs. the predictions of qppr were evaluated further with an external data set of clint for 11 vocs. the development of a global qppr model for metabolism was initially undertaken using experimental data on the in vivo intrinsic clearance of 26 vocs in rats, collated and evaluated in previous studies by bliveau et al. (1,1,1,2-tetrachloroethane ; 1,1,2,2-tetrachloroethane ; 1,1,2-trichloroethane ; 1,1-dichloroethane ; 1,1-dichloroethylene ; 1,2-dichloroethane ; benzene ; bromochloromethane ; bromodichloromethane ; carbon tetrachloride ; chloroethane ; chloroform ; cis-1,2-dichloroethylene ; dibromomethane ; dichloromethane ; ethylbenzene ; hexachloroethane ; isoprene ; methyl chloride ; m - xylene ; n - hexane ; pentachloroethane ; styrene ; toluene ; trichloroethylene ; vinyl chloride). subsequently, the ing qppr model was evaluated with experimental in vivo data on clint for 11 additional vocs in rats (1,1,1-trichloroethane ; 1,2,4-trimethylbenzene ; bromoform ; dibromochloromethane ; furan ; halothane ; o - xylene ; trans-1,2-dichloroethylene ; tetrachloroethylene ; propylene ; ethylene). these 11 chemicals outside the calibration set were also lipophilic, low - molecular - weight vocs and likely substrates of cytochrome p450 2e1. moreover except for halothane and 1,2,4-trimethylbenzene, the chemicals of the evaluation dataset possess values of pow, ionization potential, and blood: water pc within the range of values for the chemicals in the qppr calibration set. for qppr modeling, clint (expressed in units of l blood, clintblood, or l phospholipids, clintpl) initially, clintblood (l blood / h / kg) for all the studied chemicals was computed as allometrically scaled vmax (mol / h / kg)/km (mol / l blood). since cyps are located in the endoplasmic reticulum embedded in the phospholipidic bilayer, the clintpl values reflecting chemical affinity for the phospholipids (pl) were subsequently computed. the values of clintpl (l phospholipid / h / kg) were obtained by dividing vmax (mol / h / kg) with km expressed as mol / l pl. the km values in m of pl were obtained by multiplying the values of km expressed as mol / l blood with the chemical - specific phospholipid: blood partition coefficients (pplb) calculated as follows: pplb=0.3poa+0.7pwapba, where poa is the n - octanol: air pc, pwa the water: air pc, and pba the blood: air pc. the above equation computes pplb as the ratio of phospholipid: air to blood: air pcs of the vocs, based on poulin and krishnan. the input parameters required for converting the clint obtained from the literature were poa, pwa, and pba. poa and pwathe n - octanol: air pc (poa), was calculated as the product of the n - octanol: water pc (pow) and pwa (inverse of henry 's law constant at 37.5c). the n - octanol: air pc (poa), was calculated as the product of the n - octanol: water pc (pow) and pwa (inverse of henry 's law constant at 37.5c). pbaexperimental values were used for rat blood: air. the calculated values of pplb for the chemicals used for the development and for the evaluation of the qppr are reported in table 1. experimental values were used for rat blood: air. the calculated values of pplb for the chemicals used for the development and for the evaluation of the qppr are reported in table 1. the rate and affinity for p450-mediated metabolism would appear to be related to the size, shape, charge, and energy of the substrate; therefore variables that reflect these properties were chosen for the qppr analysis. the descriptors of the size and shape of the molecule were the molecular length, width, depth, volume, surface, and the kappa 2 index, as well as two descriptors used in the work of lewis et al. , namely, the ratio of the molecular length to the molecular width (l / w) and the ratio of the area of the molecule (i.e., length times width) to the square of the depth (a / d). the dipole moment and ionization potential (ip) were used as measure of the charge disposition and the energy in the molecule, respectively. the values of all the previously cited descriptors were calculated using commercially available software (molecular modeling pro, chem sw, fairfield, ca). before calculating the molecular descriptors with molecular modeling pro, the 3d molecules were drawn and minimized using the full mm2 (molecular mechanics program) method provided in the software. the dipole moment and the ionization potential hydrophobic descriptors such as log pow (log of the n - octanol : water pc) that reflect hydrogen bonding and - stacking have already been correlated to the values of metabolic constants. in this study, the following physicochemical parameters were chosen to describe the relative solubility and partitioning into diverse biological media: log pow, log phospholipid: water pc (log pplw); log blood: water pc (log pbw), and log water: air pc (log pwa). the blood: water and phospholipid: water pcs were obtained by dividing the blood: air and phospholipid: air pcs values by the water: air pc values. the values of pow, pwa, blood: air, and phospholipid: air pcs were obtained as described for the calculation of pplb. multilinear regression analysis approach was chosen for the qppr analysis of clint because linear regression models are simple, transparent, and easy to reproduce. stepwise regression analysis was performed to select the qpprs based on the most statistically significant independent variable(s) from an a priori list (see section 2.1.4). the coefficient of determination r, the adjusted r (radj ; adjusted for number of variables), the standard error of the estimate s, and the value and significance of the f statistic were calculated. the normality of the residuals was checked visually on normal probability plots of the standardized residuals (i.e., expected normal cumulative probability versus observed cumulative probability). leave - one - out cross - validation was conducted and the were expressed in terms of q, a measure of precision error of the model. the q was computed as follows: q2=1pressssy, where press is that predicted residual sum of squares and ssy the sum of squares of the response values. the statistical significance (p < 0.05) of the regression coefficients was estimated by a t statistic test. multicollinearity refers to the occurrence of correlation between two independent variables in the multiple linear regression model. multicollinearity of the variables in the model was assessed by calculating the variance inflation factor (vif) for all independent variables. the value of vif was calculated as follows: vifi=11ri2, where vifi is the variance inflation factor of the independent variable i in the multilinear regression model and ri the coefficient of determination of the regression between the independent variable i and the other independent variables in the multilinear regression model. for each model, the application domain was documented by reporting the ranges of values of the descriptors, the modeled response, and the endpoint. a qppr model was considered adequate when: the values of r and radj were 0.6, the value of q was 0.6, and the independent variables were not highly correlated (i.e., vif < 4). the predictions of the qppr model were obtained in terms of lower and upper bounds of the 95% mean confidence intervals (lmci and umci, resp .) in order to represent the uncertainty associated with the mean predicted value. the lmci and umci for the 11 vocs, not in the qspr calibration dataset, were obtained by adding them in the spss file containing the data used for the qppr, along with the values of their independent variables only. in the pbpk model, the value of intrinsic clearance was calculated as the product of the qppr value of clintpl (l of pl / h / kg) and the phospholipid: blood pc (values of pplb in table 1). the intrinsic clearance (l blood / h / kg) was used within the human pbpk models to compute the hepatic clearance. the rate of metabolism was calculated on the basis of hepatic clearance (i.e., hepatic clearance times the arterial concentration). for chloroethane, dichloromethane, vinyl chloride, and dibromomethane a first - order constant (1, 2, 1, and 0.7 h, resp .) was included in the calculation of the hepatic clearance, clh (l / h): clh = qle, where e = (clint + kf vl)/((clint + kf vl) + ql ), ql is the blood flow through the liver (l / h), clint the intrinsic clearance (l blood / h), kf the first order metabolic constant (h), and vl the liver volume (l). the qppr values of clint were included in a human pbpk model for inhaled vocs. briefly, the pbpk model consisted in four tissue compartments (i.e., liver, fat, richly, and poorly perfused tissues) and a gas exchange lung, which were interconnected by blood flows. the distribution of vocs into tissue compartments was described as perfusion limited, and the metabolism was limited to liver. to evaluate the impact of uncertainty on the metabolic rate, for all the chemicals, pbpk simulations were also conducted by setting the value of e to 0.999 (emax) and then to 0.001 (emin), respectively. the human physiological parameters of the pbpk model (i.e., body weight = 70 kg ; cardiac output = 18 l / h / kg ; alveolar ventilation = 18 l / h / kg ; tissue compartment volumes, fraction of body weight : liver = 0.026 ; richly perfused tissues = 0.05 ; poorly perfused tissues = 0.62 ; fat = 0.19 ; perfusion of the tissue compartments, fraction of cardiac output : liver = 0.26 ; richly perfused tissues = 0.44 ; poorly perfused tissues = 0.25 ; fat = 0.05) were obtained from tardif et al.. table 1 presents the value of the partition coefficients used in the pbpk model (i.e., blood : air, tissue : blood, and phospholipid : blood pcs). the phospholipid: blood pc was calculated using, whereas the blood: air pc and tissue: blood pcs were gathered from the literature. the pbpk model (differential and algebraic mass - balance equations, physiological parameters, qspr equations for metabolic constants, and pcs) was written in acsl (acslx, version 2.5, aegis technologies group, inc, huntsville, al). the model code is included in the supplementary data available online at doi:10.1155/2012/286079. to compare the impact of different (uncertain) scenarios of rate of metabolism on the pharmacokinetics in human, simulations were carried out by setting (i) the value of clint equal to the lower and upper bound of the qppr predicted mean 95% confidence interval, or (ii) the liver extraction ratio to 0.001 (no metabolism) and 0.999 (maximum extraction). the 24 h venous blood kinetics corresponding to the four scenarios of metabolism were simulated for an 8 h exposure to 1 ppm of each voc. the 24 h area under the curve (auc24) of the venous blood kinetics was also calculated to compare the four scenarios of metabolism simulated with pbpk models. additionally, the venous blood kinetics of m - xylene, toluene, ethylbenzene, dichloromethane, styrene, 1,2,4-trimethylbenzene, and 1,1,1-trichloroethane were compared to experimental data. the applicability of the qppr model was evaluated on the basis of the level of uncertainty in the qppr estimate and the impact (sensitivity) of metabolism on the auc24. figure 1 illustrates the role of uncertainty and sensitivity in the reliability of the qppr - pbpk modeling framework, based on reference. the sensitivity of the metabolism to the auc was estimated by the ratio of the auc24 obtained with no metabolism (emin) to that obtained with the maximum theoretical metabolism (emax). the sensitivity of auc24 to metabolism was considered to be low, medium, or high if the ratio (aucemin/aucemax) was within a factor of 2, within an order of magnitude, or greater. the uncertainty in the qppr prediction was evaluated by comparing it to the experimental data. the prediction uncertainty was considered to be low, medium or high if the prediction was within a factor of two, within an order of magnitude and above 10-fold of the experimental data, respectively. this approach was applied to evaluate the reliability of applying the qppr within the pbpk model for two situations: (i) for the calibration set of chemicals, for which the uncertainty of the qppr was evaluated by comparing the predictions of clintpl with the experimentally derived clintpl values and (ii) for chemicals in the evaluation dataset, for which the uncertainty in the qppr prediction was considered to be high , to replicate the data poor situations with new or tested chemicals with unknown experimental clint values. the initial effort to develop a qppr model for metabolism rate (expressed as clintblood, in units of l blood / hr), based on a stepwise analysis of its relationship to various molecular descriptors and physicochemical properties, was not successful (not shown). same analysis, repeated for clint expressed in units of l pl / h (clintpl), yielded a qppr that consisted of log pplw, log pbw, and ip (ionization potential, ev) as input parameters. this model satisfied the criteria for an acceptable model in terms of coefficient of determination (r = 0.802 ; radj = 0.775), leave - one - out cross validation (q = 0.755), and multicollinearity (vifs : log pplw = 2.42 ; log pbw = 2.38 ; ip = 1.04). the values of the regression coefficients were significant (p value < 0.001 for the constant, log pplw and log pbw, and 0.007 for ip). however, as the value of log pow can be obtained more readily than log pplw, the regression analysis was repeated by using log pow, log pbw, and calculated ip, and it yielded the following qppr: log clintpl=5.63(1.187)1.287(0.149)log pow + 1.08(0.233)log pbw 0.328(0.111)ip. this qppr model satisfied the criteria for an acceptable model in terms of coefficient of determination (r = 0.796 ; radj = 0.768), leave - one - out cross validation (q = 0.748), and multicollinearity (vifs : logpow = 2.42 ; log pbw = 2.38 ; ip = 1.04). the application domain of the model can be described with as follows: log pow =; logpbw; calculated ionization potential. the qppr was subsequently applied to calculate the clintpl of the vocs in the calibration set. table 2 presents the values of the input parameters, along with the experimental data for the 26 vocs used in qppr development. figure 2 illustrates the comparison of the predicted values of clintpl (lmci and umci) and the experimental data. the uncertainty in the predicted log clintpl can be characterized by the difference between the umci and the lmci; this value ranged from 0.37 (1,1-dichloroethane) to 1.23 (n - hexane) with a mean of 0.54 and a standard deviation of 0.18. the nearest confidence bounds of the predicted log clintpl were higher than 5-fold of the experimental value (exp .) for three substances (cis-1,2-dichloroethylene, lmci = 0.55 versus exp. = 0.09 ; styrene, lmci = 0.45 versus exp. = 0.09 ; and 1,1,2-trichloroethane, umci = 0.46 versus exp. = 0.02). the impact of the imprecision of these qppr predictions of the metabolic constants on the pharmacokinetics in humans was then evaluated by pbpk modeling. figure 3 presents the predictions of the 24 h blood pharmacokinetics following 8 h exposure to 1 ppm of each of the 26 vocs used in the qppr analysis. the bold lines represent the simulations obtained using 0 and 1 as the hepatic extraction ratio, whereas the grey area encompassed by thin lines represents the simulation obtained using lmci and umci of predicted clint in pbpk models. overall, the envelope of the concentrations predicted using the qppr predictions reduced the region of uncertainty associated with the complete lack of knowledge of hepatic extraction ratio in humans (i.e., ranging from 0 to 1). the average ratio (standard deviation) of the pbpk model simulated values of the end - of - exposure blood concentrations (i.e., cmax) obtained with emin and emax was 4.19 1.81. the lowest and highest ratios, based on the theoretical bounds of hepatic extraction (i.e., emin and emax), were observed for isoprene (1.63) and 1,1,2,2-tetrachloroethane (8.05), respectively. however, the average ratio (standard deviation) of the pbpk model simulated values of the end - of - exposure blood concentrations, based on qppr - generated bounds (lmci, umci), was 1.29 0.27. this ratio was the highest for hexachloroethane (2.39) and the lowest for 1,1-dichloroethylene (1.06). for the 26 vocs used in the development of the qppr, the values of auc24s for a 1 ppm continuous exposure the ratio of the highest to the lowest auc predicted with emin and emax was 4.3 1.94 ranging from 1.63 (isoprene) to 8.7 (1,1,2,2-tetrachloroethane). the ratio of the maximum to minimum concentrations predicted using the qppr metabolism rate was 1.36 0.4 ranging from 1.06 (1,1-dichloroethylene) to 2.8 (isoprene). figure 4 illustrates the range of predictions of venous blood pharmacokinetics compared to experimental data. overall, the predicted envelope of concentrations approximated reasonably the experimental data for dichloromethane, ethylbenzene, styrene, toluene, and m - xylene. the reliability of applying the qppr within the pbpk model was assessed for the 26 vocs in the calibration dataset (table 4). the uncertainty of the qppr prediction was estimated as the ratio of predicted clintpl to experimental clintpl. for 3 vocs (isoprene, 1,1-dichloroethylene, and vinyl chloride) the sensitivity of auc to clint was low (ratio of aucs < 2) whereas uncertainty of the clint qppr was low for isoprene and vinyl chloride and medium for 1,1-dichloroethylene. for the other 23 vocs, the ratio of aucs was between 2 and 5. for 16 of the later 23 vocs (benzene ; bromochloromethane ; bromodichloromethane ; chloroform ; dibromomethane ; 1,2-dichloroethane ; hexachloroethane ; n - hexane ; pentachloroethane ; styrene ; 1,1,1,2-tetrachloroethane ; 1,1,2,2-tetrachloroethane ; toluene ; 1,1,2-trichloroethane ; trichloroethylene ; m - xylene) the prediction uncertainty was low, thus the confidence in using the qppr in the pbpk model is high for these compounds. the uncertainty was medium for the prediction of clintpl for 7 vocs (carbon tetrachloride ; chloroethane ; 1,1-dichloroethane ; cis-1,2-dichloroethylene ; dichloromethane ; ethylbenzene ; methyl chloride). therefore, for these chemicals, the confidence in using the qppr in an inhalation pbpk model to evaluate the auc is medium. the qppr model was applied to predict the clintpl of 11 vocs that were not in the calibration dataset. table 5 presents the values of the input parameters along with the experimental data for the 11 vocs used in qppr evaluation. figure 5 illustrates the comparison of the predicted values of clintpl (lmci and umci) and the experimental data. the average difference (standard deviation) between the umci and the lmci was 0.57 0.11 ranging from 0.46 (bromoform) to 0.84 (1,2,4-trimethylbenzene). the highest umci - lmci ranges were obtained for furan (0.62), tetrachloroethylene (0.63), and 1,2,4-trimethylbenzene (0.84). the nearest predicted values of umci and lmci on log clintpl were greater than 5-fold of the experimental data for tetrachloroethylene (lmci = 0.02 versus exp = 1.8). as in the qppr development section, the impact of the imprecision on these log clint predictions on the pharmacokinetics in humans was evaluated by pbpk modeling. figure 6 presents the predictions of the 24 h blood pharmacokinetics following 8 h exposure to 1 ppm of each of the 11 vocs used in the qppr evaluation. the bold lines represent the simulations obtained using 0 and 1 as the hepatic extraction ratio, whereas the grey area encompassed by thin lines represents the simulation obtained using lmci and umci of predicted clint in pbpk models. the reduction of the region of uncertainty associated with the complete lack of knowledge of hepatic extraction ratio in humans (i.e., ranging from 0 to 1) by the envelope of the concentrations predicted using the qppr predictions was observed for the 11 vocs. the mean ratio (standard deviation) of the pbpk model simulated values of the end - of - exposure blood concentrations obtained with emin and emax was 3.92 2.13 ranging from 1.42 (ethylene) to 7.45 (bromoform). however, the same average ratio (standard deviation) of pbpk simulated blood concentrations, based on qppr - generated bounds (lmci and umci) was 1.2 0.1, ranging from 1.07 (ethylene) to 1.33 (bromoform). table 6 presents the values of the auc24s (mg / l - h) for the 11 vocs used in the evaluation of the qppr. the average ratio of the highest to lowest auc predicted using emin and emax was 4.08 2.31 (mean sd). the lowest and highest ratios, based on the theoretical bounds of hepatic extraction (i.e., e = 0.001 or 0.999), the ratio of the maximum to minimum concentrations predicted using the qppr metabolism rate was 1.2 0.1, ranging from 1.07 (propylene) to 1.33 (dibromochloromethane). figure 7 illustrates the range of predictions for two of the chemicals in the external dataset (1,2,4-trimethylbenzene and 1,1,1-trichloroethane) venous blood pharmacokinetics compared to experimental data. envelope of concentrations simulated reasonably the experimental data for 1,2,4-trimethylbenzene whereas the blood concentrations of 1,1,1-trichloroethane were underestimated by about 30%. the reliability of applying the qppr within the pbpk model was assessed for the 11 vocs in the evaluation dataset, using the framework shown in figure 1. considering that the experimental data of clintpl for new or untested chemicals will be essentially unknown, it is realistic to consider the uncertainty of the qppr prediction of clintpl to be high for all chemicals in the evaluation dataset. the of the analysis of applicability for the chemicals in the evaluation dataset are reported in table 7. for 3 vocs (ethylene ; propylene ; 1,1,1-trichloroethane) the sensitivity was low (ratio of aucs < 2) thus the reliability of using their clint qppr in the pbpk was considered high. for the other 8 vocs (bromoform ; dibromochloromethane ; trans-1,2-dichloroethylene ; furan ; halothane ; tetrachloroethylene ; 1,2,4-trimethylbenzene ; o - xylene), the ratio of the maximum to the minimum possible aucs was between 2 and 5, such that the confidence in using the qppr in an inhalation pbpk model to evaluate the aucs is medium for these chemicals. sars, qsar, qsprs, and qpprs have been developed for various toxicological and chemical properties but only very few studies have focused on developing such models to parameterize pbpk models. a limitation in developing pbpk models relates to the availability of the metabolic constants (clint, vmax, and km). quantitative relationships between structure and metabolism rates have been investigated for a limited number of closely related compounds, even though their applicability to pbpk modeling has not been demonstrated (e.g., qspr models for kcat and 1/km). other works in this area relate to the development of quantum chemical or quantum dynamic methods for prediction of activation energy or enthalpy of activation of p450 mediated reactions , which have not been used to derive metabolism constants for direct incorporation within rodent or human pbpk models. the use of the group contribution method to develop qsprs for integration within pbpk models has been successfully demonstrated, particularly for the inhalation toxicokinetics of vocs. this approach however is limited to vocs containing one or more of the molecular groups or fragments for which the contribution has been evaluated (i.e., ch3, ch2, ch, c, c = c, benzene ring, h on benzene ring, and halogens). in order to extend the applicability domain then, it is important to investigate the feasibility of developing qsprs based on more global, physicochemical properties. in this regard, the present study investigated the development of a qppr, that used chemical properties rather than chemical structure as input, and it was calibrated to predict clint expressed in terms of chemical affinity to phospholipids in the endoplasmic reticulum in which cyp enzymes are embedded. this logical transformation of clint data, reported here for the first time in literature, facilitated the development of more adequate qppr than the conventional clint based on blood concentrations. all efforts to develop qpprs for predicting clint based on blood concentrations were unsuccessful. the qppr based chemical affinity to phospholipids obtained in this study should be regarded as a screening level tool to provide plausible range of metabolism rates in order to facilitate a first - cut evaluation of the blood concentration of inhaled vocs in humans. the uncertainty associated with this qppr tool should be evaluated along with the sensitivity of clint on the dose metrics of the chemical of interest, in the perspective of intended precision. accordingly, if the dose metric is highly sensitive to clint and the qppr predictions of clint are highly uncertain, then the present tool is of limited use even for screening purposes. in such cases, then in vivo or in vitro studies can be undertaken to get chemical - specific estimates of clint. the qppr predictions were reasonably in accordance with experimental values for most but not all chemicals in the calibration and evaluation datasets. for some chemicals, the predicted values of log clint for 1,1,1-trichloroethane (figure 5(a) ) and tetrachloroethylene (figure 5(k) ) exceeded the experimental values by two orders of magnitude. the qspr for rat hepatic clearance developed by bliveau and colleagues also overestimated the metabolic rate of these two vocs. however the pbpk model for 1,1,1-trichlorethane indicated that the auc of parent chemical in venous blood is not sensitive to vmax and km. this was demonstrated in figure 6(a), showing that qppr - overestimation of clintpl of 1,1,1-trichloroethane led only to a minimal impact, in terms of the underestimation of the venous blood concentration. in the case of tetrachloroethylene, a poorly metabolized halogenated voc, the overestimation of the clintpl led to a 3-fold underestimation of the cmax (figure 6(k) ) or a 4 - 5-fold underestimation of the auc24 (table 7). if this magnitude of error is not acceptable for screening - level evaluation, then the metabolic rate should be experimentally determined. the combined assessment of the uncertainty / sensitivity of metabolic constants in pbpk models would facilitate the determination of the applicability of the qppr model, given the level of precision need for an application (figure 1) the qppr developed in this study is a generic tool to provide initial estimates of clint of vocs metabolized by hepatic cyp. it does not take into account stereochemistry or other pathway - specific rates and processes, which may be important for some chemicals (e.g., predicted values of clint are almost identical for 1,1-dichloroethylene and cis-1,2-dichloroethylene but experimental values vary by log units of 1.06). therefore, predictions of clint based on generic considerations are likely to be inaccurate for specific chemicals but are of limited use in that the estimates (along with the bounds, representing the level of uncertainty) can be integrated with human physiology to provide a first - cut view of the plausible kinetic profiles. the utility of the qppr models depends, in part, on the ability to reproducibly calculate the descriptors. hence, in this study, the descriptors that could be easily calculated and interpreted were chosen and obtained using episuite (for log pow and pwa) and mmpro (for the ionization potential). however, the blood solubility parameter (i.e., blood : air pc) is additionally required and this can either be obtained experimentally in vitro or using other qsars that account for protein (i.e., haemoglobin and plasma protein) binding in addition to solubility considerations. there are some algorithms and qsars available in this regard, but further development is necessary to adequately account for the protein binding phenomena in human blood for various classes of chemicals. the qppr developed in this study computes clintpl, which can then be converted to clintblood for use in pbpk modeling. in an effort to evaluate whether the same input parameters can be used to relate to clintblood these yielded the following equation (significant terms only): log clintblood=5.1170.305logpow0.324ip. even though and give almost identical (one for clintpl and the other for clintblood) despite the differing r values (0.796 versus 0.402), it should be noted that was obtained based on statistical analysis of calibration dataset (i.e., modeling) whereas was derived simply by fitting clintblood to the specific input parameters. further rearrangements and simplifications of the qppr, as well as the loss of accuracy associated with such attempts, were not performed in the current study. the output of the qppr developed in the present study is log clint, which is useful for simulating pharmacokinetics in humans of chemicals at low levels of exposure. clint is applicable to first - order situations (i.e., when blood levels in humans are much lower than the km for metabolizing enzyme) and is derived by dividing the vmax (i.e., the enzyme turn - over) with km (representing the affinity of the substrate for the enzyme). the input parameters of the qppr, namely, log pow and log pbw, are estimates of the relative solubility in octanol, water, and blood. then, an interpretation of the model for clint could be that the binding to the p450 enzyme is a of hydrophobic interactions which, in turn, can be estimated with parameters reflective of the solubility in n - octanol and blood. the solubility in blood is the sum of the solubility in its components (water, phospholipids, neutral lipids, and proteins). most of the studied vocs are likely to bind to hemoglobin because of their lipophilicity (log pow value above 1) and low molecular volume. the pbw, thus, is likely an indicator of the binding to proteins, whereas the log pow reflects more the affinity for biotic lipids in the metabolism microenvironment. similar to log pow, the ionization potential has already been correlated with metabolic rates, namely, the vmax and vmax/km, as this latter parameter could be correlated with the energy needed to break a covalent bond for the oxidation of the substrate. the qspr model for clint developed in this study has a defined theoretical endpoint, is nonambiguous, has a defined domain of application, was analyzed using appropriate goodness - of - fit (r) and robustness (q), and has an attempt of mechanistic interpretation. the in vivo dataset on 26 vocs used for the qppr calibration was chosen because it was previously collated and used in qspr analyses. the qspr analysis was also attempted with the entire dataset of 37 vocs (calibration + external dataset) but it did not improve the goodness - of - fit statistics (not shown). the predicted bounds of the 95% confidence interval of intrinsic clearance were incorporated within a pbpk model to predict the blood toxicokinetics of vocs. the simulations of blood kinetics were comparable to experimental data for 6 vocs (toluene, m - xylene, ethylbenzene, styrene, dichloromethane,1,2,4-trimethylbenzene, and 1,1,1-trichloroethane, figures 4 and 7). the simulations obtained in the present study, using lower and upper confidence intervals on the mean predicted clint, reduced clearly the uncertainty bounds associated with the total lack of knowledge (i.e., e ranging anywhere between 0 and 1). furthermore, the present study incorporated the qppr predictions of clint along with physiological parameters, such that impact on in vivo kinetics could be simulated. in effect, in some cases where the uncertainty on clint predictions was high, it did not translate into a proportionate error on the predictions of kinetics, due to the additional consideration of physiological constraints, and such observations are critical in data - poor situations for designing focused studies to generate chemical - specific data in vitro or in vivo. the qppr developed in this study approximated the experimental rat metabolic constants for the various low - molecular - weight vocs; and it was used along with the human physiology to generate initial or screening level values of clint to construct human pbpk models that could be of potential use to interpret data such as measured biomarker levels or for designing kinetic studies to reduce database uncertainty. as shown with some vocs (e.g., figure 3 : 1,1,1,2-tetrachloroethane, hexachloroethane, and n - hexane), the blood concentration profile is extremely influenced by clint, such that metabolism can not be neglected in simulating or interpreting human exposure data. and in such cases, the ability to generate at least a range of plausible values of clint, as done in the present study, would facilitate first in - human simulations of pharmacokinetics of parent chemicals. integrating information on the impact of metabolism on dose metrics (i.e., auc) along with prediction uncertainty of the qppr facilitates the determination of the level of confidence in using this screening level tool. depending upon the overall confidence in the qppr application for predicting dose metrics (low, medium, and high) relative to the use purposes, overall, the qppr developed in the present study allows to predict the clint of vocs on the basis of generic molecular descriptors rather than with fragment constants as done previously. the chemical concentration in phospholipids, for the first time, was found to be a dose metric amenable to qppr analysis. the qppr was then used to generate range of values of clint; the level of confidence in these estimates was assessed by considering the impact of clint on the simulated dose metrics (i.e., auc of parent chemical in venous blood). for other dose metrics and situations, a more robust qppr needs to be developed, and such efforts can be based on the methodological developments accomplished in this study. the qppr - based simulation of pharmacokinetics reduced the range of uncertainty for few substances relative to complete lack of knowledge of the clint, but it needs to be evaluated / refined with much larger dataset should this screening - level approach be adopted for providing more precise estimates of metabolism rates. overall, the integrated qppr - pbpk model developed in this study is a potentially useful tool for characterizing and reducing the uncertainty associated with the complete lack of knowledge of clint in predicting human pharmacokinetics of inhaled vocs.

the objectives of this study were (i) to develop a screening - level quantitative property - property relationship (qppr) for intrinsic clearance (clint) obtained from in vivo animal studies and (ii) to incorporate it with human physiology in a pbpk model for predicting the inhalation pharmacokinetics of vocs. clint, calculated as the ratio of the in vivo vmax (mol / h / kg bw rat) to the km (m), was obtained for 26 vocs from the literature. the qppr model ing from stepwise linear regression analysis passed the validation step (r2 = 0.8 ; leave - one - out cross - validation q2 = 0.75) for clint normalized to the phospholipid (pl) affinity of the vocs. the qppr facilitated the calculation of clint (l pl / h / kg bw rat) from the input data on log pow, log blood: water pc and ionization potential. the predictions of the qppr as lower and upper bounds of the 95% mean confidence intervals (lmci and umci, resp .) were then integrated within a human pbpk model. the ratio of the maximum (using lmci for clint) to minimum (using umci for clint) auc predicted by the qppr - pbpk model was 1.36 0.4 and ranged from 1.06 (1,1-dichloroethylene) to 2.8 (isoprene). overall, the integrated qppr - pbpk modeling method developed in this study is a pragmatic way of characterizing the impact of the lack of knowledge of clint in predicting human pharmacokinetics of vocs, as well as the impact of prediction uncertainty of clint on human pharmacokinetics of vocs.

in nearly all countries of the world, diabetes mellitus is one of the most common chronic diseases and continues to increase in numbers and significance, because of the reduced physical activity and increased obesity caused by changing lifestyles. it is estimated that the global figure of diabetes patients is about 250 million currently, and the number is expected to be 380 million by 2025. between 2010 and 2030, there will be a 69% increase in number of adults with diabetes in developing countries and a 20% increase in developed countries. in north america, about 9095% of all cases of diabetes are type 2 diabetes mellitus (t2 dm), and the population over 65 with t2 dm is about 20%. t2 dm may in severe complications, including renal failure, blindness, slow healing wounds, and arterial diseases. about 510% of the total health care budget has been used for t2 dm in many countries. in a general classification, diabetes mellitus is divided into two groups: type 1 diabetes mellitus (t1 dm) and t2 dm. t1 dm or insulin dependent diabetes mellitus (iddm) is an autoimmune disease that leads to destruction of islet beta cells in the pancreas, which in a complete halt of insulin production, while t2 dm, also called noninsulin dependent diabetes mellitus (niddm), is a heterogeneous disorder characterized by a progressive decline in insulin action (insulin resistance) in liver and peripheral tissues, accompanied by the inability of beta cells to compensate for insulin resistance (pancreatic beta cell dysfunction) leading to overt hyperglycemia. insulin resistance, characterized by reduced responsiveness to normal circulating concentrations of insulin, is a common feature of almost all patients with t2 dm, and it plays a key role at the beginning and in the development of whole process of t2 dm. the presence of insulin resistance leads to increased beta cell insulin secretion with compensatory hyperinsulinemia. impaired function of beta cell will cause deterioration in glucose homeostasis, at this point, insulin secretion can not keep pace with the underlying insulin resistance and glucose intolerance, then t2 dm occurs. in patients destined to develop t2 dm, the beta cell compensatory response declines, and then develop relative or absolute insulin insufficiency. previous studies have demonstrated that t2 dm is a multifactorial disease in which genetic factors consisting of multiple susceptibility genes and environmental factors contribute to the disease development, and the pathogenesis of t2 dm is still unclear now. because of ethical considerations, clinical studies of human diabetes, especially disease pathology research is constrained to a certain extent, thus using various animal models can induce the disease by a different mechanism but with the similar or same and offer promise of new insights into human diabetes, which is advantageous in biomedical studies owing to the uncertain etiology of t2 dm and its causes are multifarious. rodent seems to be the most suitable model for the study of t2 dm because of the small size, short generation interval, easy availability, and economic considerations. in this study , we aim to review the current available spontaneous rodent models for t2 dm with regard to their characteristic features, advantages, and disadvantages in order to investigate whether they can replicate the pathophysiological process of t2 dm. right now, there are lots of rodent models available for the study of t2 dm, but some of them may not always be satisfactory to simulate human t2 dm totally due to the large heterogeneity in the latter. obviously, no single rodent model can represent the onset and development of human t2 dm in all details. taken together, the existing rodent models provide a rich array of opportunities for investigators to study the complex pathogenesis and pathophysiological process of t2 dm. there is no fully unified classification criteria for rodent models of t2 dm, and in this paper various types of rodent models of t2 dm will be divided into spontaneously diabetic rodent models, artificially induced diabetic rodent models, and transgenic / knockout diabetic rodent models on the basis of their strains, features, advantages, and disadvantages. among these large number of animal models of t2 dm, the spontaneous type 2 diabetic rodent models are considered the most outstanding and most useful. we will respectively introduce several spontaneous rodent models of t2 dm in order to investigate the process of insulin resistance of t2 dm in human in the following part. obesity and the consequent insulin resistance are major features of t2 dm in human beings, and they play a key role at the beginning and in the development of the whole process of t2 dm. consequently, obese animal models of t2 dm have been used to simulate the complex pathogenesis and pathophysiological process of human disease and to gain insights into the human condition. the ob / ob mouse, db / db mouse, and zucker fatty (fa / fa) rat are the most typical examples of t2 dm obese models with monogenic . on the other hand, the kk mouse, the nzo mouse, the oletf rat, and the nsy mouse are the representation of obesity - induced diabetes models with polygenic . since these models show severe obesity, hyperinsulinemia, and insulin resistance throughout their lives, the agents like insulin sensitizers, antiobesity, and some other antihyperglycemia agents, which decrease the body weight and improve peripheral insulin sensitivity, have been largely tested by making use of these obese rodent models. leptin, the product of the ob gene, may be a partial factor contributing to insulin resistance. the ob / ob mouse strain, from the bar harbor - jackson laboratory, has a well - known feature of leptin deficiency because of the mutation identified in leptin gene leading to severe insulin resistance. the ob gene was transferred from the stock of origin onto the b/6 genomic and was located on chromosome 6. in early 1970s, these ob / ob mice were used to investigate the pathogenesis of insulin resistance as the first rodent model. these ob / ob mice exhibit rapid gain in body weight and may reach three times of the normal weight of wild - type controls. in addition to obesity, early in the life of these mice, insulin resistance and hyperinsulinemia occur, which are out of proportion to their adiposity at early stages. in the ob / ob model, hyperinsulinemia manifests at 3 to 4 weeks of age together with hyperphagia and insulin resistance. on the contrary, leptin treatment in ob / ob mice causes decrease of both glucose and insulin levels within hours of administration before either food intake or body weight changes. similarly, in normal rodents leptin also has a clear insulin - sensitizing effect acutely and after chronic administration. in many rodent models of obesity, increased glucocorticoids also can mediate both the hyperphagia and insulin resistance due to leptin deficiency or resistance. in ob / ob mice, at least in rodents, suppressing hypothalamic - pituitary - adreno - cortical (hpa) axis by leptin replacement may be an important component of leptin action on insulin sensitivity. the primary defect in the ob / ob mice is from neuroendocrine origin, but the exact aberration and its site are not yet fully known. its expression is a lack of satiety control at the hypothalamic and/or pituitary level. the major action site of leptin is hypothalamus, where neurons are directly regulated by leptin reside. neuropeptide y (npy), produced by hypothalamus, is a neuromodulator implicated in the control of energy balance and in the hypothalamus of ob / ob mice. overproduced as a central effector of leptin deficiency, npy is associated with obesity, a typical symptom of t2 dm and infertility, and eventually plays a major role on insulin action or secretion regulation in central nervous system (cns). the symptom of t2 dm of ob / ob mice attenuates with age, being manifested by the continuous decline of plasma insulin levels in the second year of life, with a consequent effect of glucose tolerance and insulin resistance improvement and a loss of adipose body weight. it is interesting to notice that ob / ob mice are not known to develop major diabetic complications despite the marked insulinemia in contrast to other species. the diabetes db gene mutation occurred spontaneously in the leptin - receptor - deficient c57bl / ksj strain of mice and is originally derived from autosomal recessive mutation on chromosome 4 with complete penetrance, originating from bar harbor, maine. the db / db mouse can be considered as having a natural history that closely parallels to human beings. it becomes hyperphagic, hyperinsulinemic, and insulin resistant early in life (within 2 weeks of age), then develops obesity at the age of 3 to 4 weeks. the hyperglycemia becomes manifest at the age of 4 to 8 weeks due to beta cell failure. at this time , the insulin secretion of pancreatic beta cell depletes and the hyperinsulinemia recedes, the mouse exhibits ketosis, gradual body weight loss, and deceases no longer than 8 to 10 months. in view of this, the sequence of events in this model appears to mimic human t2 dm, but it seems not compatible to investigate the complication of t2 dm because of the short life span. however, it is reported that the db / db mouse was used to investigate the renal and microvascular diabetic complications. leptin controls food intake and neuroendocrine function by activating the long form of leptin receptor and ultimately regulates adiposity which influences insulin sensitivity. it also regulates glucose homeostasis independent on the energy balance in the same way. owing to alternative splicing, the leptin receptor which belongs to the cytokine receptor class i superfamily has several well - known isoforms in mouse, while the long form lepr b isoform (lepr - b) is the only stat3 signaling - competent isoform. compared with ob / ob mouse, the db / db mouse represents similar hypothalamic disturbances and npy abnormalities as a of lack of leptin receptors. at the same time, the leptin is still produced but it can not activate the leptin receptors and restrain obesity and hyperinsulinemia induced by npy. in order to manifest this viewpoint, some investigators treated db / db mouse with exogenous leptin, the showed that leptin administration has no effect on food intake and body weight gaining. the kk mouse originating from japan is a polygenic model of obesity and t2 dm. it was crossed with the bar harbor c57bl/6j mouse, and the body size of original strain of this mouse was large. in 1957, kondo et al. reported the kk mouse at first, which spontaneously exhibited distinct adiposity, hyperglycemia, and hyperinsulinemia. at about 2 months of age, the kk mouse manifested moderate obesity due to hyperphagic, which was associated with insulin resistance, compensatory hyperinsulinemia, and islet cell hyperplasia. the insulin resistance and hyperinsulinemia reached to the peak at about 5 months and returned to normal at 9 to 12 months due to beta cell failure. the kk mouse bred several substrains throughout the world, and they vary both genetically and phenotypically from each other. among these substrains, kk / ay mouse, also named as yellow kk mouse, developed as a of the dominant mutation of yellow agouti (ay) gene in japanese kk. homozygous kk / ay mouse will die before implantation or shortly thereafter, whereas heterozygous kk / ay mouse can grow up representing severe obesity, insulin resistance, hyperglycemia, and hyperinsulinemia at the age of about 8 weeks. the hyperglycemia and hyperinsulinemia of kk / ay mouse continue to develop along with the age and it has no need to maintain a high energy diet in order to become diabetic. for the past many years , the kk / ay mouse was used as mild hyperglycemia model for studying the treatment to prevention and intervention with obesity. zucker fatty (fa / fa) rat, along with finding of the spontaneous mutation (fatty) in the rat stock of sherman and merck, was found by zucker in harriet bird memorial laboratory during 1974 and 1975. on chromosome 5 of the zucker fatty (fa / fa) rat, the simple autosomal recessive (fa) gene is mutant spontaneously, which in hyperphagia and early onset of obesity. at the age of 4 weeks , the zucker fatty (fa / fa) rat from the obese strain gained weight more rapidly because of the increased growth of subcutaneous fat depot, and it had a considerably higher body weight at about the age of 9 weeks. like the db / db mouse model, the hyperphagia and obesity in the zucker fatty (fa / fa) rat are attributed to hypothalamic defect in leptin receptor signaling, which is also associated with mild hyperglycemia, insulin resistance, mild glucose intolerance, hyperlipidemia, hyperinsulinemia, and moderate hypertension. the zucker diabetic fatty (zdf) rat was an inbred line of zucker fatty rat developing into a genetic model, which was established in 1985 and in 1991. compared with zucker fatty rats, male zdf rats become less obese but more insulin resistant, and then rapidly progress to frank diabetes because of the lack of sufficient insulin secretion required adequate compensation for the insulin resistance. the male zdf rat represents hyperglycemia at about 7 weeks of age, and fully diabetic at 12 weeks. the serum insulin levels of male zdf rat reach the peak at about 7 to 10 weeks, but as soon as the beta cells of pancreas can not respond to glucose stimulus, the insulin levels drop subsequently as pancreatic cease to. the female zdf rat develops diabetes just on a diabetogenic diet unlike male zdf rat. it has been reported that in the progression of zdf rat, the decrease of beta cell glucose transporter 2 (glut-2) membrane receptors and the concomitant loss of muscle glucose transporter 4 (glut-4) transporters are responsible for the impaired insulin secretion and subsequent hyperglycemia. the activity of glut-4 receptors decreased in adipose tissue and skeletal muscles of zdf rat, which leads to the decreased beta cell transport ability together with the peripheral insulin resistance. the decrease of glut-2 membrane receptors coupled with insulin resistance and beta cell dysfunction in zdf rat can be commonly used to simulate the mechanism of human t2 dm and test insulin sensitizers and other various agents. the new zealand strain of obese mice, a model of polygenic obesity, which was obtained by selective inbreeding from a stock colony in the hugh adam department of cancer, considerably gains weight during the first 10 weeks of life as a of hyperphagia and reachs to peak at about 12 months, coupled with the corresponding hyperglycemia and hyperinsulinemia. unlike other obese models, the nzo mouse manifests insulin resistance at an early age and gradually represents hyperleptinemia together with leptin resistance. with the growth of nzo mouse, hyperglycemia and impaired glucose tolerance increase continuously, and the level of blood glucose reaches 300400 mg / dl at the age of 20 to 24 weeks. in nzo mouse, the hyperglycemia occupies a leading position despite the fact that the level of insulin is lower than that in other obese models because of the distinct peripheral insulin resistance and increased gluconeogenesis. although nzo mouse is a rare preferred model, it becomes a useful model for studying the relationship between autoimmunity, obesity, and diabetes, and its new recombinant congenic strains are used to study the the otsuka long - evans tokushima fatty (oletf) rat was obtained by selectively inbreeding from an outbred colony of long - evans rats purchased from charles river, canada, develops mild to moderate obesity early at about 4 weeks with hyperglycemia later in life at around 18 to 25 weeks age. oletf rats exhibit the characteristics of innate polyphagia, mild obesity, late onset of hyperglycemia, hyperinsulinemia, hypertriglyceridemia, hypercholesterolemia, chronic course disease, and clinical onset of diabetes mostly in males, which are similar to the pathophysiologic progress of human t2 dm. many recessive genes on several chromosomes including the x chromosome are involved in the induction of diabetes in oletf rats. it is also observed that the oletf rats carry a null allele for the cholecystokinin a receptor which may be related to the regulation of food intake. the pancreatic islets of oletf rats change progressively, and less than 9 weeks of age, the islets just exhibit mild lymphocyte infiltration, then show hyperplastic alterations and fibrosis in or around islets at around 10 to 40 weeks of age, finally more than 40 weeks, represent atrophy of islets. with oletf rats, many investigators carried out some researches to test exogenous insulin and antidiabetic drugs such as cilnidipine, acarbose, pioglitazone, and so on. the nagoya - shibata - yasuda (nsy) mouse, an inbred polygenic animal model of t2 dm, was established by selective inbreeding from the jc1: icr mouse, from which the nod mouse was also derived. the nsy mouse closely imitates human t2 dm in which the characteristics of nsy mouse are mild obesity with abdominal and visceral fat accumulation, accompanied by impaired insulin secretion and moderate insulin resistance contributing to diabetes development in an age - dependent manner. for nsy mice , there is a marked gender difference that almost all males develop diabetes, while the percentage for females is only about 30%. the nsy mouse is also a polygenic model of t2 dm, and three major susceptibility loci (nidd1n, nidd2n, and nidd3n) have been mapped on chromosomes 11, 14, and 6. the nsy mouse is particularly useful for studying the age - related damages and phenotypes of t2 dm, as well as the undiscovered genetic differentiations and correlations between t1 dm and t2 dm. in 1992, the tsumura suzuki obese diabetes (tsod) mouse, accompanied with the other strain named tsumara suzuki non - obese (tsno), was established by tsumura and suzuki through repeatedly selective inbreeding of obese male mice of ddy strain. differing from the tsno mouse that does not become obese, the tsod mouse exhibits polygenic obesity and insulin resistant at about 2 months old, which in hyperinsulinemia and hyperglycemia, but just in males. in tsod mouse, pancreatic islets are hypertrophic and -cell mass increases, which controls the blood glucose levels so that the severe diabetes does not develop. the impaired glut4 translocation in both skeletal muscle and adipocytes of tsod mouse is one of the important reasons for the reduced insulin sensitivity and insulin resistance. it is investigated that many susceptibility loci have been mapped on chromosomes 11, 1, and 2, which are closely related to obesity, hyperglycemia, and hyperinsulinemia of tsod mouse, and the combination of these genetic loci may lead to the symptoms of tsod mouse similar to human t2 dm. m16 mice, a unique line of mice created through election for 3 to 6 weeks weight gain for many generations from an outbred icr base stock in institute of cancer research in london, are a new animal model to simulate human obesity and t2 dm. compared with icr, both males and females of m16 mice gain weight at early age and maintain moderate obesity at all ages because of hyperphagia, accompanied by increased body fat percentage, fat cell size and numbers, and organ weights. at 8 weeks of age, all m16 mice exhibit hyperglycemic, hyperinsulinemic, and hypercholesterolemic relative to icr, but the fasted blood glucose levels are, respectively, 56 percent higher in males and 22 percent higher in females. the spontaneously hypertensive obese rat (shr / ncp), an obese model of t2 dm with hypertension, part of the ant descendants exhibits obesity with normotensive or mildly hypertensive, which is determined by an autosomal recessive trait. the characteristics of shr / ncp rats are hyperphagia and early onset of obesity, insulin resistance, impaired glucose tolerance, hyperinsulinemic, and normal or slight hyperglycemia. a new model of obese type 2 diabetes, spontaneously diabetic torii (sdt) fatty (fa / fa) rat, was established by introducing the fa allele of the zucker fatty rat into the spontaneously diabetic torii (sdt) rat genome via the speed congenic method. compared to male sdt rats, sdt fatty (fa / fa) rats showed overt obesity, and hyperglycemia and hyperlipidemia at younger ages (5~6 weeks), which is associated with hyperphagia by an induced disorder of leptin action, and plasma triglyceride (tg) and total cholesterol (tc) levels in sdt fatty (fa / fa) rats were significantly higher than those in original sdt rats. furthermore, with an early incidence of diabetes mellitus, diabetes - associated complications (such as renal lesions and cataract) in the sdt fatty (fa / fa) rat are seen at younger ages than in the sdt rats. the pharmacological effects of antidiabetic drugs, such as metformin, pioglitazone, and dipeptidyl peptidase-4 inhibitor, have been tested on sdt fatty (fa / fa) rats. sdt fatty rat is expected to be a useful model for analysis of diabetic complications and the evaluation of drugs used for metabolic disease. it is demonstrated that human t2 dm can also exist in the absence of obesity. thus, it is necessary for developing nonobese models to study this condition of the t2 dm. the gk (goto - kakizaki) rat, cohen diabetic rat, and spontaneously diabetic torii (sdt) rat are the typical examples of nonobese models of t2 dm. these nonobese rodent models are very useful for studying the mechanisms of diabetes complications (e.g., renal, retinal, and peripheral nerves lesions). however, because of their absence of obesity and hypoinsulinemia, only very few studies on drug testing using these models have been reported in the literature in 1973, goto and his collaborators in japan developed the goto kakizaki (gk) rat, a polygenic nonobese model of t2 dm with early and relatively stable hyperglycemia, hyperinsulinemia, and insulin resistance, through selective inbreeding of nondiabetic wistar rats with mild glucose intolerance over many generations. for gk rats, because of the reduced number of islets at birth, both insulin resistance and impaired insulin secretion are present at their adult lives, coupled with moderate but stable fasting hyperglycemia which is present at the end of the first 2 weeks. and after 8 weeks, hyperglycemia degenerates and insulin secretion of the islets stimulated by glucose is more severely impaired, but generally, during its lifetime, fasting glucose remains mild and stable and rises only after challenge with glucose. for gk rats, 60% of pancreatic -cell mass decreased along with the distinct defects on -cell function, because of the secondary loss of -cell differentiation manifested by the three genetic loci correlated with impaired insulin secretion and glucotoxicity. in the pancreatic islets of gk rats at 2 months of age, the deposition of fibrous and nonamyloid material bring about the that the islets become transmuted in shape but do not increase in total area. with the exception of the defects of pancreatic -cell mass, the decrease of insulin sensitivity in peripheral tissues and hepatic glucose overproduction are also mechanisms of development of hyperglycemia and insulin resistance. for gk rat, developing some features of complications of diabetes can be used to study the diabetic complications seen in humans, including glomerulopathy, peripheral neuropathy, and retinopathy. the gk rat is one of the useful animal models for studying the defects of pancreatic -cell mass and pathophysiologic progress of t2 dm and its complications. cohen diabetic rat, an exceptional genetical model derived from diet - induced t2 dm model by placing the rat on a synthetic 72% sucrose - copper - poor diet for 2 months, displays many features of the human t2 dm. meanwhile, the cohen diabetic rat expresses genetic susceptibility (sensitivity and resistance) to a carbohydrate - rich diet, a central feature of t2 dm in human beings. nevertheless, the cohen diabetic rat has many shortcomings, a major of which is that it has never been systematically characterized in terms of phenotype or genotype. for this reason, the cohen diabetic rat model has been studied until recently only to a limited extent after being established nearly 30 years ago. spontaneously diabetic torii (sdt) rat is a new spontaneously nonobese diabetic strain derived from the sprague - dawley rat in 1997 at torii pharmaceutical co., ltd. it has been found that the distinct characteristics of this model are glucose intolerance, hyperglycemia, hypoinsulinemia, and hypertriglyceridemia. it was reported that there is a major cumulative incidence of diabetes in male rats (100% by 40 weeks of age) than in female rats (33.3% even by 65 weeks of age). because of the chronic severe hyperglycemia, sdt rats develop diabetic retinopathy (dr) , diabetic peripheral neuropathy , and diabetic nephropathy. with worldwide rises of t2 dm incidences, many of the animal models have been established by different mechanisms to describe similar characteristic features of this human disease and new therapeutic methods still need to be found. among these large numbers of animal models of t2 dm, the spontaneous rodent models are the most outstanding, distinctive, and useful one, because of the small size, short generation interval, easy availability and economic considerations. additionally, many of the spontaneous animal models can commendably simulate the characteristics of t2 dm, such as obesity, insulin resistance, hyperinsulinemia, hyperglycemia, and hyperlipemia. however, every spontaneous rodent model of t2 dm has either advantages or disadvantages and each of them can not replicate all characteristic features of t2 dm because of the complex, heterogeneous, multifactorial syndrome of this disease. so, in the future, more promising animal models that closely simulate human t2 dm regarding all aspects of t2 dm are needed urgently.

diabetes mellitus, especially type 2 diabetes (t2 dm), is one of the most common chronic diseases and continues to increase in numbers with large proportion of health care budget being used. many animal models have been established in order to investigate the mechanisms and pathophysiologic progress of t2 dm and find effective treatments for its complications. on the basis of their strains, features, advantages, and disadvantages, various types of animal models of t2 dm can be divided into spontaneously diabetic models, artificially induced diabetic models, and transgenic / knockout diabetic models. among these models, the spontaneous rodent models are used more frequently because many of them can closely describe the characteristic features of t2 dm, especially obesity and insulin resistance. in this paper , we aim to investigate the current available spontaneous rodent models for t2 dm with regard to their characteristic features, advantages, and disadvantages, and especially to describe appropriate selection and usefulness of different spontaneous rodent models in testing of various new antidiabetic drugs for the treatment of type 2 diabetes.

anti - n - methyl - d - aspartate receptor (anti - nmdar) encephalitis is an autoimmune limbic encephalitis induced by antibodies directed against the nr1 subunit of the nmdar. typical patients with anti - nmdar encephalitis show nonspecific prodromal symptoms, such as a fever and headache, followed by symptoms resembling schizophrenia, and then develop generalized seizure, altered mental status, hypoventilation, autonomic instability, and characteristic movement disorders, such as orofacial - limb dyskinesia and catatonia. anti - nmdar encephalitis was originally reported as a paraneoplastic syndrome associated with ovarian teratoma. however, it is now acknowledged that the spectrum of this encephalitis is much broader, as there have been many cases in women without ovarian teratoma, men, and children. there is also a possibility that pregnancy and/or delivery could trigger anti - nmdar encephalitis, as several patients developed this disorder during pregnancy or in the postpartum period. we herein report a japanese patient who developed severe anti - nmdar encephalitis three weeks after normal delivery and discuss the pathophysiology of postpartum anti - nmdar encephalitis. the patient was a 24-year - old primiparous japanese woman with no significant medical history. she had no complications during the course of the pregnancy and gave birth to a healthy baby girl via vaginal delivery. one week later, she presented with auditory hallucination and abnormal behavior and was mandatorily hospitalized in the department of psychiatry of a general hospital. she was diagnosed with postpartum psychosis and treated with antipsychotic drugs. on the second hospital day, she presented with somnolence and unstable breathing followed by generalized seizure. on the third hospital day, she developed status epilepticus and hyperthermia and was transferred to the intensive care unit. generalized seizure was difficult to control despite treatment with propofol and antiepileptic drugs, and respiratory depression led to tracheal intubation and artificial ventilation. she was treated with methylprednisolone (mpsl) pulse therapy at a dose of 1 g for 3 days and intravenous immunoglobulin therapy (ivig) at a dose of 0.4 g / kg for 5 days (fig . 1). however, her symptoms deteriorated gradually and she developed involuntary movements in the face and right upper limb. simv: synchronized intermittent mandatory ventilation, ps: pressure support, cpap: continuous positive airway pressure, ivig: intravenous immunoglobulin, pe: plasma exchange, dfpp: double filtration plasmapheresis, mpsl: methylprednisolone, dvt: deep vein thrombosis on admission, her body temperature was 38.5c. a neurological examination showed orofacial dyskinesia and athetoid movement in the right hand even under deep sedation with propofol. laboratory tests revealed inflammatory reaction (white blood cell, 13,350/l ; c reactive protein, 4.31 mg / dl) and mild liver dysfunction (aspartate aminotransferase, 37 iu / l ; alanine aminotransferase, 103 tests for herpes simplex, herpes zoster, and epstein - barr virus were negative . autoantibodies were all negative, except for anti - thyroglobulin antibody and anti - thyroperoxidase antibody . the of a cerebrospinal fluid ( csf) analysis showed lymphocytic pleocytosis (82/l, mononuclear cells 77/l), a slightly elevated protein level (51 mg / dl), and a normal glucose level (73 mg / dl). anti - nmdar antibody was positive (20, examined by cosmic corporation, tokyo, japan) in the csf. electroencephalogram (eeg) demonstrated diffuse beta activity superimposed on frontally dominant high - voltage rhythmic delta bursts, consistent with 2 ). brain magnetic resonance imaging (mri) showed slightly increased signal intensity with swelling in the bilateral medial temporal lobes on t2 and flair imaging (fig . abdominal computed tomography ( ct) revealed a right ovarian cystic tumor with small calcifications (fig . 4a). based on the characteristic clinical findings and positivity for anti - nmdar antibody, a diagnosis of anti - diffuse beta activity superimposed on frontally dominant high - voltage rhythmic delta bursts was observed. brain magnetic resonance image (mri) of the patient (flair image, axial view). (a) mri performed at 1 month after onset. high signal intensity and slight swelling were seen in the bilateral medial temporal lobes. 4b ) consisting of stratified squamous epithelium with cutaneous appendage, neural tissue with choroid plexus, adipose tissue, bone, cartilage, and intestinal structure with goblet cells. in addition to resection of the teratoma, she was treated with ivig, mpsl pulse therapy, plasma exchange (pe), and double filtration plasmapheresis (dfpp); however, her involuntary movements, higher brain dysfunction, and autonomic dysfunction were prolonged. we could not perform further intensive immunosuppressive therapy, such as rituximab and cyclophosphamide, due to repetitive severe infections, including pneumonia and sepsis (fig . after the fifth ivig, her involuntary movements and respiratory failure improved gradually, and she was transferred to rehabilitation hospital eight months after onset ( fig . 1). at the time of transfer, she was still in a bedridden state and had severe cognitive dysfunction. brain mri 8 months after onset showed an almost normal appearance with no abnormal signals or brain atrophy. her neurological condition continued to improve after transfer, and she achieved independent gait and verbal communication at 12 months after onset. neither brain atrophy nor abnormal signals were revealed on mri (fig . 3b), but her memory disturbance and mental juvenility persisted even two years after onset. (a) computed tomography of the pelvis showed a cystic tumor (1.5 cm in maximal diameter) with small calcifications adjacent to the right ovary. ovarian teratoma is the most common associated tumor of anti - nmdar encephalitis in female patients and contains antigenic neural tissue. in addition, the majority of patients have a history of prodromal flu - like symptoms, including headache, fever, nausea, vomiting, diarrhea, or upper respiratory tract symptoms. therefore, the combination of ectopic expression of nmdar, especially the nr1 subunit contained in the teratoma, and the adjuvant effect of the prodromal flu - like syndrome is thought to contribute to initiation of the immune response and production of pathogenic antibodies. the present case involved an ovarian teratoma with the development of anti - nmdar encephalitis three weeks after delivery without prodromal flu - like symptoms. therefore, anti - nmdar encephalitis may have been triggered by normal vaginal delivery in our patient. with regard to the relationship between anti - nmdar encephalitis and pregnancy / delivery, 10 patients developed anti - nmdar encephalitis during pregnancy, and 5 patients developed the disease during the postpartum period. one of the major immunological modifications during pregnancy is the th1/th2 shift, due to the progressive increases in levels of progesterone and estrogen during pregnancy, which suppress th1 cytokines and stimulate th2-mediated immunological responses as well as antibody production. therefore, th1-mediated diseases, such as rheumatoid arthritis and multiple sclerosis, tend to improve during pregnancy and worsen during the postpartum period. in contrast , th2-mediated diseases, such as systemic lupus erythematosus, tend to worsen during pregnancy. however, recent studies have shown that annualized relapse rate of neuromyelitis optica (nmo) tends to decrease in the first half of pregnancy and increase after the pregnancy, although nmo is a th2-mediated disease. in myasthenia gravis, worsening of symptoms is more likely during the first half of pregnancy and postpartum. therefore, the influence of pregnancy and delivery on patients with immunological disorders should be analyzed with respect to each disease. with regard to anti - nmdar encephalitis , the precise immunopathogenesis remains to be elucidated, although b cells, but not t cells, have been proposed to be involved. therefore, further investigations regarding the immunopathogenesis of the disease, including the th1/th2 balance and cytokine dynamics, and large - scale epidemiological surveys are necessary to determine the effects of pregnancy and delivery on anti - nmdar encephalitis. several hypotheses have been proposed to explain the pathomechanism of anti - nmdar encephalitis associated with pregnancy and/or delivery. it was demonstrated that markedly increased estrogen promotes formation of autoreactive lymphocytes through rapid maturation of b cells and secretion of interleukin-10 during pregnancy, which may facilitate the production of anti - nmdar antibody and/or its crossing of the blood - brain barrier, thereby contributing to the development of anti - nmdar encephalitis. indeed, symptomatic recovery seemed to accelerate after giving birth in three patients with anti - nmdar encephalitis. in contrast, the maternal immune system is modified to allow immune tolerance to fetal antigens during pregnancy. yu et al. hypothesized that pregnancy - induced modulation of the immune system may contribute to the occurrence of postpartum anti - nmdar encephalitis by a breakdown in tolerance to self - antigens. table shows the clinical characteristics of patients with anti - nmdar encephalitis that developed during the postpartum period. all of the patients showed psychotic symptoms, including anxiety, delusions, bizarre behavior, insomnia, agitation, irritability, hallucinations, psychomotor excitement, confusion, and depression, and were suspected of having postpartum psychosis in the early period of the encephalitis. the patient reported by koksal et al. showed dramatic improvement shortly after teratoma resection. in contrast, the patient reported by yu et al. showed little improvement following tumor resection, mpsl, and pe, but improved dramatically shortly after rituximab administration. rituximab was also very effective in a patient with postpartum anti - nmdar encephalitis without teratoma. our patient showed very slow improvement after tumor resection, which may have been caused by the delay in surgical resection and hesitation to perform aggressive immunosuppressive treatment, such as rituximab, due to recurrent severe bacterial infections. severe bacterial infections and deep vein thrombosis are complications which occur frequently in patients with severe anti - nmdar encephalitis. management of these complications is crucial and has a considerable effect on the prognosis of anti - nmdar encephalitis. ivm: involuntary movement, gtcs: generalized tonic - clonic seizure, rt.: right, mpsl: methylprednisolone therapy, pe: plasma exchange, ivig: intravenous immunoglobulin therapy, dfpp: double filtration plasmapheresis to our knowledge, there are no obvious differences in the clinical characteristics of patients between anti - nmdar encephalitis associated with pregnancy, associated with delivery, and with no pregnancy / delivery association. recently screened 96 consecutive patients with postpartum psychosis and 64 healthy postpartum women and found 2 patients with postpartum psychosis positive for anti - nmdar antibody. both patients recovered after treatment with lithium, lorazepam, and antipsychotic agents, and remission was sustained, despite the absence of any immunosuppressive treatment, suggesting that mild anti - nmdar antibody encephalitis may be underdiagnosed among a cohort of postpartum psychosis patients.

we describe a 24-year - old woman with anti - n - methyl - d - aspartate receptor (anti - nmdar) encephalitis that developed 3 weeks after normal delivery. she was treated with methylprednisolone, intravenous immunoglobulin, and plasmapheresis, in addition to teratoma excision. however, her recovery was slow, and dysmnesia and mental juvenility persisted even two years after onset. to date, five patients with postpartum anti - nmdar encephalitis have been reported. all of those patients showed psychotic symptoms and were suspected of having postpartum psychosis in the early period of the encephalitis. changes in hormonal factors, modification of immune tolerance, or retrograde infection of the ovary may be contributing factors for postpartum anti - nmdar encephalitis.

to identify genes with sex - limited and nonsex - limited functions, we searched flybase (tweedie et al . 2009) for mutations within the following phenotypic categories: visible, lethal, semilethal, sterile, male sterile, and female sterile (searches used the termlink section ; http://flybase.org/static_pages/termlink/termlink.html). we trimmed the data set to include alleles associated with specific genes (although many alleles have been mapped to specific chromosomes and/or cytological bands, the mapping resolution for these cases was generally insufficient to be included within the final data set). individual genes can potentially have multiple alleles within the data set (though the majority of drosophila genes were not associated with any alleles). we therefore classified each gene according to its range of mutant allele phenotypes, which fall between entirely female - specific to entirely male - specific. the genes were classified as follows: genes with female - limited fitness effects are those that contain female - sterile alleles and no other allele type;genes with female - biased fitness effects contain female - sterile alleles and any combination of visible, lethal, and semilethal alleles;genes with male - limited fitness effects contain male - sterile alleles and no other allele type;genes with male - biased fitness effects contain male - sterile alleles and any combination of visible, lethal, and semilethal alleles;genes without sex - biased fitness effects contain both male - sterile and female - sterile alleles, visible alleles, lethal alleles, and/or semilethal alleles. genes with female - limited fitness effects are those that contain female - sterile alleles and no other allele type; genes with female - biased fitness effects contain female - sterile alleles and any combination of visible, lethal, and semilethal alleles; genes with male - limited fitness effects contain male - sterile alleles and no other allele type; genes with male - biased fitness effects contain male - sterile alleles and any combination of visible, lethal, and semilethal alleles; genes without sex - biased fitness effects contain both male - sterile and female - sterile alleles, visible alleles, lethal alleles, and/or semilethal alleles. genes associated with sterility, but with neither sex specified (the underlying allelic data did not provide information about sex), were considered ambiguous and excluded from the analysis. the sample of sterile alleles that were included in the analysis is potentially heterogeneous because some studies examine fertility in only one sex rather than both. nevertheless, the proportion of sex - limited and nonsex - limited steriles in our data set is consistent with independent experimental that explicitly test male and female fertility (alleles associated with sex - specific sterility are roughly three times as common as alleles associated with sterility in both sexes ; see lindsley and lifschytz 1972 ; ashburner et al . this suggests that most genes and alleles classified as sex - limited are in fact associated with sex - limited sterility . molecular expression profiles were obtained from the sex bias database ( sebida version 2.0 : http://141.61.102.16:8080/sebida/index.php ; gnad and parsch 2006). we downloaded male versus female expression ratios (m / f) from 15 different microarray studies (data were originally reported in : parisi et al . ; gibson et al . 2004 ; stolc et al . 2004 ; mcintyre et al . 2006 ; goldman and arbeitman 2007 ; ayroles et al . 2009) and m / f ratios from a meta - analysis of several studies (details of the meta - analysis are described at sebida). m / f ratios can potentially range from zero to infinity, with male - biased transcription for m / f > 1 and female - biased transcription for m / f < 1. to impose symmetry on sex - biased expression levels, we rescaled the data using an index of sex - biased expression: x = m/(m + f). this variable ranges between zero and one, with female - biased transcription for x < 0.5 and male - biased transcription for x > 0.5. the final data set included 2,433 genes with m / f expression information from at least 1 of the 15 studies. within the final data set, there were 1,955 genes with similar mutational effects on both sexes, 298 genes with female - biased fitness effects, 43 female - limited genes, 87 genes with male - biased fitness effects, and 50 male - limited genes (an additional 53 genes had ambiguous sex - specific sterility phenotypes). supplementary table s1 (supplementary material online) provides a breakdown of the data set into phenotypic subcategories, including the mean and median number of alleles per gene, per phenotypic category. whitney u tests (implemented in r ; r development core team 2005) were used to assess whether the distribution of sex - biased transcription levels differs between phenotypically defined gene categories. to examine whether different categories of sex - biased transcription have different compositions of phenotypes , we subdivided the data set into five expression categories, each with equal range: 0 < x < 0.2; 0.2 < x < 0.4; 0.4 < x < 0.6; 0.6 < x < 0.8; and 0.8 < x < 1.0. two - tailed fisher s exact tests were used to examine whether female - biased transcription categories were enriched for genes with female - specific phenotypes and whether male - biased transcription categories were enriched for genes with male - specific phenotypes. the presented below use meta - analysis expression profiles (from sebida ; see above) to transcriptionally categorize genes. the meta - analysis data set represents a composite of several independent microarray studies, which minimizes the likelihood of sex - biased transcription misclassification for each gene (compared with classifications based on single studies). the meta - analysis also includes data for a high proportion of the 2,433 genes (compared with single studies), which maximizes statistical power. the are consistent across studies, though the statistical power is often lower, due to decreased gene representation. for each platform are presented within the supplementary figs. s1 and s2 (supplementary material online). to identify genes with sex - limited and nonsex - limited functions, we searched flybase (tweedie et al . 2009) for mutations within the following phenotypic categories: visible, lethal, semilethal, sterile, male sterile, and female sterile (searches used the termlink section ; http://flybase.org/static_pages/termlink/termlink.html). we trimmed the data set to include alleles associated with specific genes (although many alleles have been mapped to specific chromosomes and/or cytological bands, the mapping resolution for these cases was generally insufficient to be included within the final data set). individual genes can potentially have multiple alleles within the data set (though the majority of drosophila genes were not associated with any alleles). we therefore classified each gene according to its range of mutant allele phenotypes, which fall between entirely female - specific to entirely male - specific. the genes were classified as follows: genes with female - limited fitness effects are those that contain female - sterile alleles and no other allele type;genes with female - biased fitness effects contain female - sterile alleles and any combination of visible, lethal, and semilethal alleles;genes with male - limited fitness effects contain male - sterile alleles and no other allele type;genes with male - biased fitness effects contain male - sterile alleles and any combination of visible, lethal, and semilethal alleles;genes without sex - biased fitness effects contain both male - sterile and female - sterile alleles, visible alleles, lethal alleles, and/or semilethal alleles. genes with female - limited fitness effects are those that contain female - sterile alleles and no other allele type; genes with female - biased fitness effects contain female - sterile alleles and any combination of visible, lethal, and semilethal alleles; genes with male - limited fitness effects contain male - sterile alleles and no other allele type; genes with male - biased fitness effects contain male - sterile alleles and any combination of visible, lethal, and semilethal alleles; genes without sex - biased fitness effects contain both male - sterile and female - sterile alleles, visible alleles, lethal alleles, and/or semilethal alleles. genes associated with sterility, but with neither sex specified (the underlying allelic data did not provide information about sex), were considered ambiguous and excluded from the analysis. the sample of sterile alleles that were included in the analysis is potentially heterogeneous because some studies examine fertility in only one sex rather than both. nevertheless, the proportion of sex - limited and nonsex - limited steriles in our data set is consistent with independent experimental that explicitly test male and female fertility (alleles associated with sex - specific sterility are roughly three times as common as alleles associated with sterility in both sexes ; see lindsley and lifschytz 1972 ; ashburner et al . this suggests that most genes and alleles classified as sex - limited are in fact associated with sex - limited sterility . molecular expression profiles were obtained from the sex bias database ( sebida version 2.0 : http://141.61.102.16:8080/sebida/index.php ; gnad and parsch 2006). we downloaded male versus female expression ratios (m / f) from 15 different microarray studies (data were originally reported in : parisi et al . ; gibson et al . 2004 ; stolc et al . 2004 ; mcintyre et al . 2006 ; goldman and arbeitman 2007 ; ayroles et al . 2009) and m / f ratios from a meta - analysis of several studies (details of the meta - analysis are described at sebida). m / f ratios can potentially range from zero to infinity, with male - biased transcription for m / f > 1 and female - biased transcription for m / f < 1. to impose symmetry on sex - biased expression levels, we rescaled the data using an index of sex - biased expression: x = m/(m + f). this variable ranges between zero and one, with female - biased transcription for x < 0.5 and male - biased transcription for x > 0.5. the final data set included 2,433 genes with m / f expression information from at least 1 of the 15 studies. within the final data set, there were 1,955 genes with similar mutational effects on both sexes, 298 genes with female - biased fitness effects, 43 female - limited genes, 87 genes with male - biased fitness effects, and 50 male - limited genes (an additional 53 genes had ambiguous sex - specific sterility phenotypes). supplementary table s1 (supplementary material online) provides a breakdown of the data set into phenotypic subcategories, including the mean and median number of alleles per gene, per phenotypic category. two - tailed mann whitney u tests (implemented in r ; r development core team 2005) were used to assess whether the distribution of sex - biased transcription levels differs between phenotypically defined gene categories. to examine whether different categories of sex - biased transcription have different compositions of phenotypes , we subdivided the data set into five expression categories, each with equal range: 0 < x < 0.2; 0.2 < x < 0.4; 0.4 < x < 0.6; 0.6 < x < 0.8; and 0.8 < x < 1.0. two - tailed fisher s exact tests were used to examine whether female - biased transcription categories were enriched for genes with female - specific phenotypes and whether male - biased transcription categories were enriched for genes with male - specific phenotypes. the presented below use meta - analysis expression profiles (from sebida ; see above) to transcriptionally categorize genes. the meta - analysis data set represents a composite of several independent microarray studies, which minimizes the likelihood of sex - biased transcription misclassification for each gene (compared with classifications based on single studies). the meta - analysis also includes data for a high proportion of the 2,433 genes (compared with single studies), which maximizes statistical power. the are consistent across studies, though the statistical power is often lower, due to decreased gene representation. for each platform are presented within the supplementary figs. s1 and s2 (supplementary material online). genes with sexually dimorphic phenotypic effects tend to have relatively sex - biased mrna expression levels (fig . 1 ; supplementary figs . s1 and s2, supplementary material online). compared with genes with similar phenotypic effects in both sexes (those with visible, lethal, semilethal, and/or alleles for sterility in both sexes), genes with female - limited and female - biased fitness effects have higher mrna expression in females (mann whitney u tests : female - limited effects genes associated with male - limited and male - biased fitness effects have higher mrna expression in males ( male - limited effects these largely extend to comparisons between individual phenotypic categories and the distribution of sex - biased expression throughout the entire drosophila genome . genes with greater phenotypic effects in females show greater female - biased mrna expression ( female - limited effects : p = 4.38 10 ; female - biased fitness effects : p < 2.2 10). genes with male - limited phenotypic effects are more male biased in transcription (p = 6.78 10), though genes with male - biased fitness effects do not differ from the genome - wide distribution (p = 0.228). unexpectedly, genes that similarly affect both sexes (e.g., lethal, visible, male, and female sterile) are relatively female biased (transcriptionally) compared with the genomic distribution (fig . each of these is highly consistent across individual microarray studies ( supplementary figs . s1 and s2, supplementary material online). genes with sexually dimorphic mrna transcription levels are associated with sex - biased fitness effects. the upper panel shows the cumulative distribution for sex - biased transcription among five phenotypic categories (color coded) and for the entire genome (the black curve). each phenotypic category differs significantly from the genome - wide distribution (mann whitney u ; p < 10) except for genes with male - biased fitness effects (p = 0.228); each category significantly differed from the nonsex - biased phenotypic class of genes (partially male - limited : p = 0.0105 ; other categories : p < 0.00001). the lower panel shows the proportion of each phenotypic class within five sex - biased transcription categories. two - tailed fisher exact tests (* * * p < 0.001 ; * * * * p < 0.0001) were used to determine: 1 ) whether female - biased genes (0 < x < 0.2 ; 0.2 < x < 0.4) were enriched for female - biased or female - limited phenotypic effects and 2 ) whether male - biased genes were enriched for male - biased or male - limited phenotypic effects (0.6 < x < although these patterns validate the intuition that male - biased genes should be more important for male fitness and female - biased genes should be more important for female fitness, such associations are far from absolute . genes with highly dimorphic transcription often have similar mutational effects on both sexes ( fig . 1, within the upper and lower 20 percent tails for x : 60 percent of female - biased genes and 38 percent of male - biased genes had roughly equal fitness effects in both sexes ; also see supplementary fig . genes with more moderate sex - biased transcription patterns are even less likely to have sex - limited fitness consequences . for example, among genes with 1.5-fold to 4-fold differential expression between the sexes ( ranges 0.2 < x < 0.4 and 0.6 < x < 0.8), roughly 80 percent had similar mutational effects in each sex. thus, although genes with male - biased and female - biased transcription are statistically associated with male - biased and female - biased fitness effects (respectively), the vast majority of sex - biased genes appear to be functionally important for both sexes (genome - wide, over 90 percent of genes fall within the range 0.2 < x < 0.8). there are several evolutionary routes that may lead to sex - biased gene expression (ellegren and parsch 2007), yet only some are expected to generate associations between the relative transcription level of a gene and its fitness consequences in each sex. sex - biased gene expression may be directly selected for when males and females have different gene expression optima. because direct selection for expression dimorphism is not necessarily expected to alter the relative importance of the gene for either sex, sex - biased mutational effects may not accompany sex - biased transcription. alternatively, the evolution of sex - biased expression may coincide with sex - specific selection for novel protein coding sequences or the differential incorporation of proteins into sex - specific genetic interaction networks (arnold et al . 2009). this may involve sex - specific expression of previously noncoding sequence, gene duplication and sex - specific cooption of paralogs, or a change in a gene s function (or subfunction) within one sex but not the other. any of these processes can generate an association between sex - biased expression and sex - biased function and phenotypic consequence. the indicate that male - biased and female - biased transcription does not necessarily equate with sex - specific importance or function. phenotypically characterized and mapped mutations tend to similarly affect both sexes, despite their variable associations with sexually dimorphic gene transcription. female - biased genes can even produce alleles that are more harmful to males than to females and vice versa, although such cases are rare. however, despite this broad pattern of shared importance, there is also a clear statistical association between transcriptional dimorphism and the probability that a gene has a sex - limited or sex - biased phenotypic effect. this implies that a fraction of male- and female - biased genes adopt sex - specific functions or become incorporated into sex - specific molecular pathways. this pattern is particularly striking when one considers the coarseness of the transcriptional data for each gene. the ratio of male to female gene expression is a composite signal from multiple tissues during the adult life - history stage and ignores the fine - scale spatial and temporal resolution that defines the process of drosophila development (meisel 2011). the association between sexually dimorphic gene expression and sex - biased phenotypic effects proves that such transcriptional assays represent a biologically and evolutionarily meaningful measurement. these patterns may also have implications for debates over the population genetic consequences of sex - specific selection (e.g., chippindale et al . 2008 ; whitlock and agrawal 2009 ; agrawal 2011 ; connallon et al . 2010 ; mallet and chippindale 2011). theory predicts that stronger selection in males than females will enhance purifying selection and reduce the mutational load of females (whitlock and agrawal 2009), whereas stronger purifying selection in females and sexually antagonistic selection between the sexes increases the female mutational load (day and bonduriansky 2004 ; bonduriansky and chenoweth 2009). considering patterns of selection across the genome, net fitness costs to females can potentially emerge when female - biased purifying selection or sexual antagonism operates across a relatively small fraction of the genome (connallon et al . the presented here suggest that, while a proportion of the genome is subject to much stronger selection in females than males, most genes with female - biased expression experience selection in males . this may either mitigate the female genetic load or expand the sequence space that can experience sexually antagonistic selection . molecular evolutionary contrasts between genes in different phenotypic categories, including selection parameter estimates from resequencing data ( using approaches described by keightley and eyre - walker 2010), may shed additional light on the population genetic consequences of sex - specific selection. one surprising observation is that alleles with similar effects on both sexes are associated with female - biased transcription, compared with the genomic distribution (fig . 1 ; fig . s1, supplementary material online). this phenotypic category is dominated by lethal alleles, which suggests that female - biased genes (at least those with moderately higher expression in females than males) have a higher proportion of essential functions than male - biased genes. these relatively severe mutational effects might partially explain why male - biased genes evolve more rapidly than female - biased genes (e.g., zhang et al . 2006 ; clark et al . 2007 ; ellegren and parsch 2007 ; larracuente et al . 2008). female - biased genes might have a smaller fraction of effectively neutral mutations or a higher degree of pleiotropy (mank et al . 2008 ; mank and ellegren 2009 ; meisel 2011), leading to decreased opportunities for neutral and adaptive evolution (e.g., fisher 1958 ; kimura 1983). these should be considered tentative, as they may be sensitive to characteristics of the available data. lethal and sterile alleles reduce fitness to zero, whereas visible and semilethal alleles may have similarly strong effects on total fitness (e.g., due to strong mate discrimination against carriers of visible and/or subviable alleles ; grossfield 1975 ; hollis et al . 2009). because mutations with small fitness effects preclude direct laboratory measurement, a broader analysis of the sex - specific fitness consequences of sex - biased genes will require a different empirical approach perhaps one that combines gene functional assays with molecular population genetics.. finally, phenotypic assays of mutations are not systematic, as they represent a collection of alleles characterized during the history of drosophila genetics research. future studies that systematically characterize genes and mutant phenotypes using targeted mutagenesis (e.g., p element insertions) or deletion mapping will further illuminate the connection between molecular and phenotypic sexual dimorphism. supplementary figures s1s2 and table s1 are available at genome biology and evolution online (http://www.gbe.oxfordjournals.org/).

genome - wide mrna transcription profiles reveal widespread molecular sexual dimorphism or sex - biased gene expression, yet the relationship between molecular and phenotypic sexual dimorphism remains unclear. a major unresolved question is whether sex - biased genes typically perform male- and female - specific functions (whether these genes have sex - biased phenotypic or fitness consequences) or have similar functional importance for both sexes. to elucidate the relationship between sex - biased transcription and sex - biased fitness consequences , we analyzed a large data set of lethal, visible, and sterile mutations that have been mapped to the drosophila melanogaster genome. the data permitted us to classify genes according to their sex - specific mutational effects and to infer the relationship between sex - biased transcription level and sex - specific fitness consequences. we find that mutations in female - biased genes are (on average) more deleterious to females than to males and that mutations in male - biased genes tend to be more deleterious to males than to females. nevertheless, mutations in most sex - biased genes have similar phenotypic consequences for both sexes, which suggests that sex - biased transcription is not necessarily associated with functional genetic differentiation between males and females. these have interesting implications for the evolution of sexual dimorphism and sex - specific adaptation.

morvan's syndrome is a rare autoimmune disorder characterized by peripheral nerve hyperexcitability, autonomic dysfunction, and central nervous system symptoms. the syndrome of muscle twitching, dysautonomia, insomnia, and delirium was first reported by morvan by the name of la choree fibrillare in 1890. we report a rare case of contactin - associated protein - like 2 (caspr2), a subtype of voltage - gated potassium channel (vgkc) complex antibody positive morvan's syndrome, with syndrome of inappropriate antidiuretic hormone secretion (siadh). a 45-year - old male presented with 4-month duration of nonradiating mild back pain, followed a month later by burning sensation in palms and soles with nocturnal exacerbations. he became aggressive, over - talkative, and insomniac over 15 days before presentation. electromyography showed spontaneous activity including myokymic discharges , doublets, and triplets in both upper and lower limb muscles. the patient had positive serum anti - caspr2 antibody, a subtype of vgkc complex detected by immunofluorescence method. his cerebrospinal fluid examination showed raised proteins 76 mg / dl (normal : 2040 mg / dl), with normal cell count (cells : 3/mm, all lymphocytes). computed tomography (ct) of the chest showed no evidence of thymoma. the patient was diagnosed as morvan's syndrome with positive anti - caspr2 vgkc antibody. spontaneous activity in right tibialis anterior showing myokymic discharges (sweep speed : 0.1 ms and sensitivity : 50 v) there was persistent low serum sodium in the range of 125130 meq / l, for which patient was evaluated. his urinary osmolarity was raised (216.36 mosm / kg, normal < 100 mosm / kg) and random urinary sodium was increased (42 mmol / l, normal < 30 mmol / l). the serum osmolarity was decreased (271.5 mosm / kg) and urinary specific gravity was 1.010. the patient was treated with intravenous immunoglobulin (iv ig) 2 g / kg in 5 divided doses. he was given phenytoin at dose of 100 mg three times a day for symptomatic relief for twitching, which acts as membrane stabilizer. the patient was started on oral prednisone (1 mg / kg) and fluid restriction was advised. he had marked improvement in muscle twitching and was able to sleep properly with immunotherapy. electromyography done 2 weeks after the course of iv ig showed decrease in spontaneous activity; occasional fasciculations were seen. his hyponatremia was also corrected. on follow - up, after 3 months, the patient was completely normal and electromyography showed no spontaneous activity. oral prednisone was given 1 mg / kg for 3 months and later tapered gradually over next 2 months. morvan's syndrome is characterized by myokymia associated with muscle pain, excessive sweating, weight loss, hallucinations, sleep disorders, and behavioral abnormality. the basic mechanism for both presentations is same, that is anti - vgkc antibody, acts at different levels of neuraxis, both at central and peripheral level. caspr2 antibodies positive cases but common in anti - leucine - rich glioma inactivated-1 (lgi-1) antibodies positive cases. lgi-1 antibodies are usually associated with hyponatremia, and caspr2 antibodies are usually associated with thymomas which carry poor prognosis. caspr2 antibodies mostly bind the neuropil, whereas antibodies to lgi-1 bound to neuronal cell bodies including the antidiuretic hormone - secreting and orexin - secreting hypothalamic neurons present in hypothalamus, raphe nucleus, and locus coeruleus. hyponatremia is not commonly reported in morvan's syndrome although it is present in half of the patient in lgi-1 antibodies positive limbic encephalitis. in addition, fasciculation, doublets, triplets, multiplets, and positive sharp waves are also present. this is a rare case report of morvan's syndrome with siadh having anti - caspr2vgkc antibodies that responded to immunosuppression.

morvan's syndrome is a rare autoimmune disorder characterized by triad of peripheral nerve hyperexcitability, autonomic dysfunction, and central nervous system symptoms. antibodies against contactin - associated protein - like 2 (caspr2), a subtype of voltage - gated potassium channel (vgkc) complex, are found in a significant proportion of patients with morvan's syndrome and are thought to play a key role in peripheral as well as central clinical manifestations. we report a patient of morvan's syndrome with positive caspr2anti - vgkc antibody having syndrome of inappropriate antidiuretic hormone as a cause of persistent hyponatremia.

benzophenone (bz4, figure 1) is a water - soluble uvb filter (max in methanol 285 nm) manufactured by basf under the trademark uvinul ms40 and used in cosmetic formulations at concentrations up to 5% (as free acid). because of its highly acidic character, it must be neutralized with sodium hydroxide or triethanolamine prior to addition to a cosmetic formulation. due to its good water and alcohol solubility, it is commonly used in aqueous preparations (shampoos, oil - free sunscreen sprays) or to protect cosmetic products including perfumes against photodegradation. benzophenone-4 was quantified in cosmetic products mainly by rp - hplc or, less frequently, si (sequential injection) analysis or ce (capillary electrophoresis). liquid chromatography was also used in bz4 quantification in environmental samples or in skin permeation studies. in such studies, a radioanalytical method involving bz4 labeled with radioisotope c (at carbonyl group) was also applied. the objective of this study was to propose a cheap and convenient method of quantification of benzophenone-4 in aqueous cosmetic preparations by hydrophilic interaction thin - layer chromatography followed by densitometry and, alternatively, by uv spectrophotometry. ethyl acetate, ethanol, methanol, potassium phosphate monobasic, and sodium phosphate dibasic were from polskie odczynniki chemiczne (poch), gliwice, poland. hair shampoos containing benzophenone-4 and benzophenone - free (blank shampoo) were purchased locally. all shampoos analyzed throughout this study were preserved with sodium benzoate, bronopol, or thiazolinone derivatives and contained sodium laureth sulfate, cocamide dea, dyes (ci 19140, ci 42080, or ci 17200), vitamins, fragrances, and plant extracts (chamomile, lavender, cornflower, and wild rose). uvinul ms40, 500 mg, was weighed accurately into a 100 ml volumetric flask, dissolved in 16.2 ml of 0.1 mol l aqueous naoh, and diluted to volume with water to give a stock solution i of the concentration 5 mg ml. the appropriate volumes of bz4 stock solution were pipetted to 25 ml volumetric flasks, ph 6 phosphate buffer was added (2 ml), and water was added to volume to furnish standard solutions of bz4 (0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, and 2.0 mg ml, bz4 expressed as free acid). 2 ml of bz4 stock solution i was diluted to 100 ml with water to furnish a stock solution ii (0.1 mg ml). the appropriate volumes of stock solution ii were transferred to 25 ml volumetric flasks, ph 6 phosphate buffer (5 ml) was added, and the solutions were diluted to volume with water to furnish solutions of bz4 of concentrations 0.004, 0.008, 0.016, 0.024, 0.032, and 0.040 mg ml (bz4 expressed as free acid). 15 mg bz4 was weighed accurately into 10 ml volumetric flasks and diluted with ph 6 phosphate buffer (1 ml) and water to volume. 0.52.5 mg bz4 was weighed accurately into 100 ml flasks and diluted with ph 6 phosphate buffer (5 ml) and water to volume. thin - layer chromatography was performed on 10 10 cm hp quality silica gel 60 plates (layer thickness 0.2 mm) from merck or on 10 20 cm standard quality silica gel 60 plates (layer thickness 0.25 mm), also from merck. plates were developed with methanol - dichloromethane 1: 1 (v / v) and dried at room temperature overnight prior to use. standard solutions prepared for method i (section 2.1) were spotted with the desaga as30 sampler equipped with a 10 l syringe (1 l spot), 15 mm from the from the plate bottom edge, and at 8 mm intervals, starting 10 mm from the plate edge and developed with ethyl acetate - ethanol - water - ph 6 phosphate buffer 15: 7: 5: 1 (v / v / v / v) as mobile phase. plates were developed in a vertical chromatographic chamber lined with filter paper and previously saturated with the mobile phase vapor for 20 min. after development, plates were dried at room temperature (20c), scanned, and analyzed in reflectance mode with the desaga cd 60 densitometer at 285 nm (rf = 0.78). spectroscopic measurements were performed with lambda 25 uv / vis spectrophotometer, perkin - elmer. (method ii) were placed in 1 cm quartz glass cuvettes and scanned over the wavelength range 200420 nm. the shampoo solutions in buffered water (method i) prepared as described above (section 2.2) were spotted on silica gel 60 hptlc plates (110 l spot depending on the expected bz4 concentration). the plates were then chromatographed as described above for bz4 standards (section 2.3). the shampoo solutions in buffered water (method ii) prepared as described above (section 2.2) were placed in 1 cm quartz glass cuvettes and scanned with the lambda 25 uv / vis spectrophotometer as described above (section 2.4 .). the sun - care preparations analyzed in this study contained benzophenone-4 and surfactants, plant extracts, preservatives, and other ingredients that may potentially absorb light within the same range as bz4. on the basis of our earlier research, it was decided that silica gel 60 is the stationary phase of choice and the most effective mobile phase capable of bz4 separation from other cosmetic ingredients was ethyl acetate - ethanol - water - ph 6 phosphate buffer 14: 7: 5: 1 (v / v / v / v). analytical wavelength suitable for bz4 analysis (285 nm) was selected on the basis of multiwavelength scans obtained for this sunscreen (figure 2). the cosmetic preparations under investigation contained raw materials other than bz4 (such as cosmetic dyes) that may absorb uv light within the same range as bz4, thus influencing the accuracy of the analysis (see figure 3, curves 1 and 2, solutions of a typical shampoo matrix containing 0% bz4). one of the most convenient methods used to reduce matrix effects in uv / vis spectrophotometry is derivative spectroscopy with the 2nd derivative being the most effective tool capable of matrix effects suppression. after the analysis of 2nd derivative spectra for bz4 standards and the shampoo matrix (figure 4), it was decided that the analytical wavelength of choice is in this case 285 nm (at this wavelength, the 2nd derivative of absorbance for the shampoo matrix is very close to 0 even at higher concentrations and for bz4 samples and standards there is a clear maximum of the da / d curve). identity of chromatographic spots for bz4 isolated from the shampoo by thin - layer chromatography was tested by comparison of their densitograms with those obtained for the bz4 standard solutions (figure 5). further investigations of the identity and purity of spots were based on the analysis of the uv spectra of bz4 isolated from the shampoo and the bz4 standards. spectra were collected directly from the chromatographic plates in the reflectance mode (for each spot three spectra were collected, for the spot center and both edges ; in figure 6 only two spectra are shown for each spot for the sake of clarity). the densitometric calibration plot was obtained by plotting peak areas against the amount of bz4 over the range 0.12.0 g spot. the densitometric calibration plot was finally generated in the form of the second - degree polynomial (table 1) and its quality was assessed by means of r values and nonnumerical analysis of residues according to (figure 7). the spectrophotometric calibration plots were linear within the studied concentration range (table 1). the general method for linearity testing proposed in this reference is based on the analysis of the ratio of the analytical signal (y) to the analyte concentration (x). if the signal - concentration relationship is linear, y / x is constant (within an acceptable range, usually 5%). when this approach was applied to the linear calibration plots generated during this study, all values of signal - to - concentration ratio were within acceptable limits for particular plots (45.54 3.3% absorbance and 23.85 2.2% for the 2nd derivative of absorbance, resp .). repeatability of chromatographic method was tested according to by replicating the entire method (section 2.5) on the same day, using the same cosmetic preparations, batches of solvents, and chromatographic plates, by the same analyst (day 1, analysis a and analysis b). intermediate precision was verified according to by repeating the procedure on the same cosmetic preparations but on a different day, by a different analyst, using other batches of solvents and chromatographic plates (day 2). the of these experiments (table 2) prove that the methods precision is sufficient for routine product analysis. the instrumental detection limits (idl) and instrumental quantification limits (iql) for chromatographic method were determined experimentally on the basis of signal - to - noise (s / n) ratio according to. blank solutions of ph 6 phosphate buffer containing zero concentration of bz4 were analyzed in triplicate along with the series of bz4 standards according to the procedure described in section 2.3. the idl and iql were calculated as the concentrations that yielded peaks with s / n of 3 and 10, respectively. the of these determinations expressed as the amount of bz4 per spot are given in table 1. in order to estimate the method loq, the minimum concentration of bz4 that can be realistically quantified in the actual cosmetic preparations, the blank shampoo samples were fortified with bz4 at the following concentrations: 0.01, 0.02, 0.04, and 0.06% (w / w), diluted with ph 6 phosphate buffer and analyzed in triplicate as described in section 2.5. in order to avoid excessive viscosity and foaming of the sample solutions, their maximum concentration was 100 mg ml and due to the capacity of the applicator syringe, the maximum application volume was 10 l. as the of these experiments, it was estimated that the lower quantification limit for bz4 in shampoos by thin - layer chromatography / densitometry without samples preconcentration is ca. instrumental limits of detection / quantification (idl and iql) were calculated from the signal - to - noise ratio (table 1) and the limit of quantification (loq) for the entire 2nd derivative spectroscopic method was studied on the basis of the same shampoo samples containing 0.01, 0.02, 0.04, and 0.06% bz4 (w / w). samples were diluted with ph 6 phosphate buffer and water (10 mg ml, higher concentrations should be avoided because of increasing matrix effects) and the estimated loq in shampoos was ca. after due consideration of factors that can influence the analysis , it was concluded that the critical points are the quality of chromatographic plates (hptlc versus tlc), the method of spotting, and the ph of the mobile phase. the same cosmetic preparations were analyzed on hptlc silica gel 60 chromatographic plates with automatic spotting and on standard tlc silica gel 60 plates with manual spotting with a hamilton microsyringe. the mobile phase ethyl acetate - ethanol - water - ph 6 phosphate buffer 15: 7: 5: 1 (v / v / v / v) was replaced with ethyl acetate - ethanol - water 15: 7: 6 (v / v / v). the of these analyses (table 2) are similar but coefficients of variations are, as it may have been expected, slightly higher for manual spotting. blank shampoo was spiked with bz4 at five concentrations: 0.5, 1.0, 1.5, 2.0, and 5.0% (w / w) corresponding to 0.25, 0.50, 0.75, and 1.0 g spot (1 l spot of the shampoo solution prepared according to section 2.2, 50 mg of shampoo per 1 ml of sample solution) and 1.25 g spot (1 l spot of the shampoo solution prepared according to section 2.2, 25 mg of shampoo per 1 ml of sample solution), respectively. the chromatographic procedure described in section 2 was performed on the samples and the recoveries are presented in table 3. spectrophotometric determination of bz4 in the same spiked shampoos was performed according to section 2.6 (sample solutions were prepared according to section 2.3, 2.5 or 1.0 mg of shampoo per 1 ml of sample solution) and the are given in table 3. standard solutions of bz4 used in this investigation were refrigerated between the experiments and not exposed to light except for time needed for plates spotting. the stability of all solutions was in these conditions excellent over at least two weeks. benzophenone-4 may be effectively separated from other cosmetic ingredients (dyes, preservatives, vitamins, and plant actives) by hydrophilic interaction high performance thin - layer chromatography on silica gel 60 with ethyl acetate - ethanol - water - ph 6 phosphate buffer as mobile phase. densitometric quantification of bz4 at 285 nm is fast, reliable, and cost - effective and it may be recommended for routine analysis of shampoos containing bz4 at different levels of concentrations (concentrations of the shampoo solutions and volumes per spot may be adjusted to the needs over a relatively broad range of bz4 concentration in a cosmetic formulation). the of chromatographic / densitometric determination (precision, accuracy) are superior to those of spectrophotometric analysis by zero derivative spectroscopy and similar to those obtained by 2nd derivative spectroscopic method but the flexibility of the chromatographic method is higher; the matrix effects do not pose a problem and the limit of quantification is slightly lower than that of the spectrophotometric analysis.

benzophenone-4 (bz4) was separated from surfactants, dyes, preservatives, and other components of hair shampoos by thin - layer chromatography on silica gel 60 stationary phase, with ethyl acetate - ethanol - water - ph 6 phosphate buffer (15 : 7 : 5 : 1 v / v / v / v) as mobile phase. densitometry scanning of chromatograms was performed at 285 nm. the densitometric calibration curve for bz4 was nonlinear (second - degree polynomial), with r > 0.999. the limits of detection and quantification were ca. 0.03 and ca. 0.1 g spot1, respectively. the obtained by hptlc - densitometry were compared to those obtained by zero and 2nd derivative uv spectrophotometry. in the case of spectrophotometric methods, calibration curves were linear with r > 0.9998. the chromatographic method was fully validated.

regular exercise is fundamental for the prevention and management of insulin - resistant disorders such as type 2 diabetes mellitus (t2 dm) and avd. while exercise affects numerous organ systems, there is increasing evidence that acute and chronic exercises have important immunomodulatory properties which may contribute to the beneficial effects of exercise on metabolism and cardiovascular health. in addition, aerobic exercise can alter the number and the function of immune cells in innate and adaptive immunities and reduce the levels of proinflammatory markers. numerous reports have demonstrated that immune cells play a critical role in the development of insulin resistance. hevener et al. showed that deletion of peroxisome proliferator - activated receptor gamma (ppar) in monocytes in glucose intolerance and decreased insulin signaling in the skeletal muscle, liver, and adipose tissue of mice. another study demonstrated that regulatory cd4 t cells expressing the transcription factor foxp3 (treg cells) are predominately found in the abdominal fat of normal mice but are reduced in the abdominal fat in mouse models of insulin resistance. this study also showed that interleukin-10 (il-10), produced by these foxp3 treg cells, inhibited tumor necrosis factor - alpha (tnf)-induced expression of proinflammatory mediators and insulin resistance in 3t3-l1 adipocytes. taken together, these findings suggest that immune cells have the potential to affect whole body glucose homeostasis and insulin action. in addition to their role in insulin resistance, immune cells are also involved in the development of atherosclerosis. in t2 dm patients, atherosclerosis is systemic, rather than localized to the heart, and also affects the microvasculature of the limbs, kidneys, eyes, and other tissues. monocytes play crucial roles in the early events that lead to this systemic atherosclerosis. yet, the molecular mechanisms that promote accelerated atherosclerosis in type 2 diabetic subjects are not fully understood. accumulating evidence implicates toll - like receptor (tlr) 2 and 4 in the pathogenesis of atherosclerosis and insulin resistance. for example, monocytes from type 2 diabetic subjects have elevated surface protein content of tlr2 and tlr4. moreover, deletion of tlr2 or tlr4 in rodents attenuates atherosclerosis and insulin resistance. in view that immune cells have a critical role in the pathophysiology of insulin resistance and avd, in this study, we examined the molecular mechanism underlying the anti - inflammatory effect of aerobic exercise on immune cells of type 2 diabetic individuals. although some studies have shown that either aerobic or resistance exercise induces a decrease in tlr4 expression in monocytes from healthy normal subjects , it is unclear whether aerobic exercise can modulate proinflammatory signaling pathways in immune cells of insulin - resistant subjects. it is well documented that aerobic exercise can improve insulin sensitivity in type 2 diabetic patients. however, no study has described whether the improvement of insulin sensitivity is due to a decrease in proinflammatory signaling pathways in immune cells of type 2 diabetic subjects. we focused our analysis on pmnc because of the considerable evidence indicating that these inflammatory cells infiltrate insulin - sensitive tissues (skeletal muscle, adipocytes, and liver) where they promote insulin resistance and are also involved in the pathophysiology of avd. therefore, we hypothesized that aerobic exercise downregulates proinflammatory signaling pathways in pmnc of insulin - resistant individuals. we studied 17 lean, 8 obese nondiabetic, and 11 obese type 2 diabetic participants. three t2 dm subjects were newly diagnosed, and one t2 dm subject was diagnosed with diabetes 2 months prior to the study. two t2 dm subjects took a sulfonylurea, which was stopped 2 days before any study procedure. all participants were sedentary (zero or one exercise bout per week) and had stable body weight (1 kg) for three months before the study. each participant underwent a medical history, physical examination, and a 75 g oral glucose tolerance test (ogtt). lean and obese participants did not have a family history of type 2 diabetes and were normal glucose tolerant. no participant was taking any medication known to affect glucose metabolism (other than sulfonylureas). the study was approved by the institutional review board of the university of texas health science center at san antonio, and all participants gave written consent. plasma glucose, insulin, and nonesterified fatty acids (nefa) were measured 30, 15, and 0 minutes before and every 15 min for 2 h after the ingestion of 75 g of glucose. within 37 days after the ogtt, vo2peak was determined using a cycle ergometer and a metabolic measurement system (sensormedics, savi park, ca, usa) as previously described. within 1 - 2 weeks after the baseline vo2peak measurement , participants returned to the clinical research center at 07:00 am to undergo a 180 min euglycemic - hyperinsulinemic (160 mu m min) clamp study as previously described. within one week of the insulin clamp, participants undertook a supervised exercise program of cycle ergometer exercise for 40 min per day, for 15 consecutive days. the 40 min of exercise consisted of 4 identical 10 min periods comprised of 8 min of exercise at 70% vo2peak followed by 2 min at 90% of vo2peak. each of these 10 min sets was followed by 2 min of complete rest. we employed this program because, during pilot experiments, we observed increases in vo2peak of ~20%. blood samples were collected immediately before and at the end of the pre- and postexercise insulin clamps for isolation of pmnc. louis, mo, usa ) density gradient, followed by centrifugation at 800 g for 30 min at room temperature. mononuclear cell (1 10cells) samples were lysed in ice - cold lysis buffer (ph 7.4), and western blot analyses were performed as previously described. after blocking with bovine serum albumin, the membranes were incubated overnight with primary antibody against tlr4 (santa cruz biotechnology, santa cruz, ca, usa) and tlr2 (ebioscience, san diego, ca, usa), which are cell surface receptors that signal through erk and c - jun amino - terminal kinase (jnk). we also performed western blotting for phosphorylated erk (invitrogen, carlsbad, ca, usa), erk (cell signaling, danvers, ma, usa), phosphorylated jnk (cell signaling, beverly, ma, usa), and jnk (cell signaling, beverly, ma, usa). bound primary antibodies were detected with a secondary antibody (anti - rabbit immunoglobulin - horseradish peroxidase - linked antibody) using enhanced chemiluminescence reagents. bands were quantitated with imagequant (ge healthcare, piscataway, nj, usa). nfb p65 binding was measured using an elisa kit (active motif, carlsbad, ca, usa), as previously described. plasma insulin (diagnostic products, los angeles, ca, usa) was measured by radioimmunoassay. glucose was measured by the glucose oxidase method using a beckman glucose oxidase analyzer, and hemoglobin a1c (hba1c) was measured using a dca2000 analyzer (bayer, tarrytown, ny, usa). plasma nefa concentrations were determined using a colorimetric method (wako, richmond, va, usa). high - sensitivity - c - reactive protein (hs - crp) was measured by elisa (alpco diagnostics, salem, nh, usa). endothelin-1 and soluble intercellular adhesion molecule-1 (sicam-1) plasma levels were measured by elisa (r&d systems, minneapolis, mn, usa). baseline comparisons between groups were done using one - way anova, and the effect of exercise training and insulin was analyzed using anova with repeated measures followed by the tukey test. sigmastat version 3.5 (systat software, san jose, ca, usa) was used for statistical analysis. table 1 shows the characteristics of study participants before and after the 15-day aerobic exercise training program. compared with the lean group, the obese and type 2 diabetic subjects had higher body mass index (bmi) and body weight. the obese group tended to have elevated plasma nefa concentrations compared with the lean group (p = 0.056). both diabetic and obese subjects had higher fasting insulin levels when compared with the lean group. the type 2 diabetic patients had higher levels of the inflammatory and endothelial markers hs - crp, endothelin-1, and sicam-1. diabetic and obese individuals were insulin resistant compared to the lean controls, based on the lower total glucose disposal during the insulin clamp. after the exercise training, the insulin - resistant individuals (obese and t2 dm groups) did not show a significant decrease in bmi or weight. similarly, there was no change in hba1c, nefa, glucose, and insulin plasma levels in the obese and diabetic groups. exercise also did not decrease the plasma levels of hs - crp, endothelin-1, and sicam-1. after exercise, vo2peak increased by 26% (p < 0.05), 14% (p = 0.08), and 8% (p the baseline m levels were 10.9 0.7, 7.8 0.8, and 4.2 0.6 mg / kgmin in lean, obese, and type 2 diabetic individuals, respectively . after exercise, insulin sensitivity ( m values) increased by 11% (p < 0.05) in lean, 15% (p < 0.05) in obese, and 32% (p < 0.05) in type 2 diabetes groups. compared with lean participants, baseline tlr4 protein content was significantly increased in the pmnc of diabetic subjects by 4.2-fold (p < 0.05) (figure 1). in obese individuals, tlr4 also tended to be elevated by 2.7-fold (p = 0.07) compared to that of the individuals in the lean group (figure 1). in contrast to tlr4, the protein content of tlr2 was not different in lean, obese, and diabetic subjects (figure 2). as shown in figure 3, baseline erk phosphorylation was significantly elevated in the diabetic subjects. the increase in erk phosphorylation was mainly observed with erk2 (3.0-fold versus the lean group ; p < 0.05). erk2 phosphorylation tended to be decreased in type 2 diabetic subjects after exercise (p = ns). we performed these measurements just before the end of the 180 min clamp, based on previous studies that showed that insulin activation of erk occurs as late as 100240 min during a clamp. it is possible, however, that insulin could have activated erk if measurements had been done at earlier time points. nfb p65 dna binding was not different between lean, obese, and type 2 diabetic participants, and neither exercise nor insulin affected nfb dna binding (figure 5). exercise training studies and cross - sectional comparisons between physically active and inactive individuals have shown a reduction in the inflammatory response to lps and lower tlr4 cell surface expression in monocytes from physically active subjects. because immune cells play a role in the regulation of glucose metabolism , we examined whether exercise improves insulin sensitivity by inducing modifications in the pro - inflammatory signaling pathways of the pmnc of insulin - resistant subjects. we anticipated that short - term training would improve insulin sensitivity in insulin - resistant individuals and that this would then be associated with a decrease in tlr4 protein content and erk signaling in pmnc. yet, acute aerobic exercise, which improved insulin sensitivity, did not affect tlr4 protein content, although it tended to attenuate erk2 phosphorylation. it is possible, however, that the 15-day intervention was not sufficient to significantly alter tlr4 protein content and erk phosphorylation. on the other hand, others have reported that a single session of cycle exercise (1.52 h long) is sufficient to decrease tlr4 cell surface expression in monocytes from healthy individuals, although tlr - downstream signaling (erk) was not examined in that study. the apparently discrepant between the present and previous studies may be related to differences in the duration of training, the type of training being performed (aerobic, resistance, or combination), the intensity of the training (moderate versus high), and/or the population examined. timmerman et al. tested the effect of combined aerobic and resistance training on monocyte subpopulations and found that tlr4 expression was reduced in proinflammatory (cd1416) but not in classical proinflammatory monocytes adhere more avidly to activated endothelial cells and are precursors to the cd16 macrophages which are distributed throughout atherosclerotic lesions. we can not rule out that the exercise intervention employed in the present study could have led to changes in tlr4 protein content and erk signaling in specific monocyte subpopulations. in this study , we found that insulin - resistant individuals have elevated tlr4 protein content in pmnc. our findings are in agreement with previous studies where tlr levels were found to be increased in inflammatory cells from type 1 and type 2 diabetic subjects, although, in contrast to the present study, subjects had increased levels of both tlr2 and tlr4. the physiologic relevance of the increased tlr4 protein content is underscored by studies in which tlr4 was ablated specifically in hematopoietic cells from mice, an intervention which reduced inflammatory markers in liver and adipose tissue and ameliorated high - fat diet - induced insulin resistance. moreover, our also demonstrate that the upregulation in tlr4 protein levels is accompanied by an increase in erk phosphorylation, especially erk2. studies conducted in ob / ob - erk1 double knockout mice showed that erk deletion improves insulin sensitivity in skeletal muscle and reduced liver fat content. defining the role of erk2 on insulin resistance future studies of tissue - specific erk2 ablation will help to clarify its role in the pathogenesis of insulin resistance and type 2 diabetes. avd is the major cause of mortality in subjects with type 2 diabetes and insulin resistance. atherosclerosis occurs earlier in the natural course of diabetes and is more severe and generalized than in nondiabetic patients. to test whether tlr4 has a direct role in the pathogenesis of atherosclerosis, michelsen et al. crossed apolipoprotein e (apoe) deficient mice, which are prone to develop atherosclerotic lesions, with mice that are null for tlr4. this group demonstrated that mice deficient in both tlr4 and apoe have a significant reduction in the size of the atherosclerotic lesions, as well as a lower number of macrophages infiltrating these lesions, compared with mice deficient with apoe only. more recently, it was reported that hematopoietic cell ablation of tlr4 reduces arterial wall macrophage infiltration and atherosclerotic lesion areas. erk also has a crucial role in the early steps in the pathogenesis of atherosclerosis because activation of erk leads to increased monocyte proliferation, migration, and differentiation into macrophages and mediates monocyte chemotactic protein-1-induced monocyte adhesion. indeed, (monocyte - derived) dendritic cells from patients with acute coronary syndrome in addition, a recent study demonstrated that hypertensive patients have increased erk phosphorylation in isolated white blood cells compared to healthy normotensive individuals, suggesting that erk activation in leukocytes may serve as a biomarker for hypertension. collectively, the from these studies and our present findings strongly suggest that enhanced signaling through tlr4 and erk, in inflammatory cells (monocytes / macrophages), plays an important role in the pathogenesis of atherosclerosis. tlr4 protein content and erk signaling were elevated in the type 2 diabetes group, which were slightly but significantly older than the lean and obese groups. based on previous reports from human and animal studies , we believe that the increase in tlr4 protein content and erk signaling is not due to the small difference in age. in fact, stewart et al. compared tlr2 and tlr4 cell surface expression on monocytes from older adults (age 6085) with that from young adults (age 1835) and found no independent effect of age on tlr2 and tlr4 levels and lps - induced cytokine production. furthermore, animal studies evaluating tlr levels and signaling in macrophages of old mice have yielded inconsistent findings. a study performed in young and old mice found that aged mouse macrophages have lower tlr4 protein. in contrast, others have not observed differences in tlr4 cell surface expression in macrophages from aged versus young mice. in this study, we did not observe differences in jnk phosphorylation between groups. in contrast, studies in mice suggest that jnk plays an important role in the pathogenesis of obesity and insulin resistance. to the best of our knowledge, most of the human studies supporting the role of jnk in insulin resistance have examined adipose tissue. moreover, our finding is in line with that of a previous study showing that jnk activity in pmnc was not associated with impaired insulin action, whereas increased jnk activity in adipose tissue directly correlated with measurements of insulin resistance. studies of nfb activity in inflammatory cells from insulin - resistant subjects have yielded contrasting . in line with a study by patel et al. , we did not observe differences in nfb activity in pmnc in insulin - resistant subjects. in contrast, ghanim et al. and dasu et al. reported increased nfb activity in pmnc from obese individuals and monocytes (a subpopulation of pmnc) from type 2 diabetic subjects, respectively. studies carried out in larger cohorts of subjects might help to clarify whether nfb signaling is up regulated in pmnc from obese and type 2 diabetic individuals and whether this alteration is specific to monocytes. in summary, acute aerobic exercise improved insulin sensitivity and cardiovascular fitness in insulin - resistant subjects. however, exercise was not associated with changes in tlr4 protein content and erk signaling in immune cells. it remains to be determined whether longer training protocols or different modes of exercise (i.e., resistance training or combination of aerobic and resistance training, as well as moderate- versus high - intensity exercise program) affect tlr4 expression and erk signaling in immune cells of insulin - resistant individuals.

. exercise has an anti - inflammatory effect against, and immune cells play critical roles in the development, of insulin resistance and atherosclerotic vascular disease (avd). thus, the goal of this study was to determine whether exercise improves insulin sensitivity in insulin - resistant subjects by downregulating proinflammatory signaling in immune cells. methods. seventeen lean, 8 obese nondiabetic, and 11 obese type 2 diabetic individuals underwent an aerobic exercise program for 15 days and an insulin clamp before and after exercise. peripheral mononuclear cells (pmnc) were obtained for determination of toll - like receptor (tlr) 2 and 4 protein content and mitogen - activated protein kinase phosphorylation. . compared with that in lean individuals, tlr4 protein content was increased by 4.2-fold in diabetic subjects. this increase in tlr4 content was accompanied by a 3.0-fold increase in extracellular signal - regulated kinase (erk) phosphorylation. exercise improved insulin sensitivity in the lean, obese, and type 2 diabetes groups. however, exercise did not affect tlr content or erk phosphorylation. . tlr4 content and erk phosphorylation are increased in pmnc of type 2 diabetic individuals. while exercise improves insulin sensitivity, this effect is not related to changes in tlr2/tlr4 content or erk phosphorylation in pmnc of type 2 diabetic individuals.

a 73-year - old man who had been transferred to our emergency room due to sudden chest pain was diagnosed with st - segment elevation myocardial infarction (stemi). primary percutaneous coronary intervention was performed. a long, white object which looked like a parasitic worm was retrieved via intracoronary aspiration and revascularization was successfully completed. contrast computed tomography revealed a huge 76 cm mass in the right upper pulmonary lobe with direct pulmonary vein invasion. histopathologic examination of the aspirated coronary object revealed pleomorphic lung carcinoma. this is an unusual case of stemi caused by lung tumor embolization via direct pulmonary vein invasion to the left side of the heart.

paclitaxel, the first microtubule stabilizer identified from the taxus brevifolia, is one of the most successful anticancer drugs currently used in the clinic. members of a second class of plant - derived microtubule stabilizers, the taccalonolides, have been isolated from a variety of tacca species. the taccalonolides are highly acetylated hexacyclic steroid lactones that exhibit effects similar to other microtubule stabilizers in that they increase the density of cellular microtubules, interrupt mitotic progression, and consequentially lead to the apoptosis of cancer cells. despite their low antiproliferative potencies in vitro, taccalonolides a and e were found to be potent and effective antitumor agents in vivo with the ability to circumvent multiple mechanisms of drug resistance, including mutations in the taxane binding site and the expression of mrp7, iii - tubulin, and p - glycoprotein (pgp). the recent isolation of taccalonolides with low nanomolar potency facilitated biochemical and structural studies demonstrating that the taccalonolides bind to tubulin covalently and impart unique interprotofilament stability to microtubules. the most potent of these rare natural taccalonolides, af, contains an epoxy group at c-22,23 that in a 231-fold increase in potency compared with taccalonolide a, which contains a double bond at this site. taccalonolide af is an effective antitumor agent that causes tumor regression in the mda - mb-231 breast cancer xenograft model, albeit with a narrow therapeutic window. a simple and efficient method was developed to semisynthetically epoxidize the c-22,23 double bond in taccalonolides a and b to generate taccalonolides af and aj, respectively. taccalonolide aj has an ic50 value of 4.2 nm, which is 734-fold more potent than the parent molecule, taccalonolide b, suggesting that epoxidation of the c-22,23 double bond is an effective way to increase the potency of this class of molecules. in the current study, the c-22,23 double bond in an additional 10 rare natural microtubule stabilizing taccalonolides was epoxidized, including the newly isolated taccalonolide ai. our demonstrate that this modification increases the potency of each taccalonolide, in some cases leading to subnanomolar potency. y, were previously isolated from various tacca sp. by multiple investigators. we have continued to search for additional rare natural taccalonolides and conduct semisynthetic reactions to fully understand the sar of this class of compounds with the goal of identifying taccalonolides with optimal properties for consideration for clinical development. the potent semisynthetic 4 is the c-22,23 epoxidation product of the naturally occurring 3. in the process of isolating highly purified 3 from the roots and rhizomes of t. chantrieri its molecular formula (c35h48o11) was determined by high resolution electrospray ionization mass spectroscopy (hresims), 645.3268 (calcd for c35h49o11 645.3269), and nmr data. the h nmr spectrum of 5 showed characteristics of the taccalonolide backbone, including four methyl singlets at 1.67 (s, 3h), 1.36 (s, 3h), 0.82 (s, 3h), and 0.76 (s, 3h), one methyl doublet at 0.95 (d, j = 7.0 hz, 3h, 21-ch3), one acetyl at 2.08 (s, 3h), epoxyl signals at 3.55 (t, j = 4.0 hz, 1h, h-2) and 3.40 (br, 1h, h-3), and an olefin singlet at 5.02 (br, 1h, h-22). the upfield shift of h-15 at 4.38 (ddd, j = 9.5, 8.3, 2.5 hz, 1h) indicated a 15-oh similar to 3 and 6. the only acetoxy group was assigned to c-12 due to the chemical shift of h-12 at 4.99 (t, j = 2.8 hz, 1h) and the hmbc correlation between h-12 and the acetoxy carbon at 169.3. the downfield chemical shift of h-1 at 4.59 (d, j = 5.2 hz, 1h) required an acyloxy substitution at c-1. this acyloxy group was determined to be an isovaleryloxy by the h nmr signals at 2.18 (m, 2h, h-2), 2.14 (m, 1h, h-3), 1.01 (d, j = 7.0 hz, 3h, h-4), 1.00 (d, j = 6.0 hz, 3h, h-5), and 2d nmr correlations. the hmbc correlations between h-1 and the carbonyl carbon of the isovaleryloxy at 171.8 confirmed this substitution. structures of taccalonolides af, a, b, aj, ai, and n. compound 5 retains the microtubule stabilizing activity of other taccalonolides and has antiproliferative activity with an ic50 of 47 nm in hela cells, which makes it one of the most potent natural taccalonolides isolated to date (table 1). the potency of this taccalonolide is consistent with previously determined sar, which indicates that a large bulky group at c-1 is optimal for activity. the bulky isovaleryloxy group at c-1 is the only difference between 5 and 6, which has an acetoxy at this site and is approximately 200-fold less potent (table 1). additionally, the only difference between 5 and taccalonolide am is the absence of the c-5 hydroxyl in 5, which confers a 42-fold increase in potency (table 1). therefore, the combination of modifications at c-1 and c-5 appear to be important for the potency of 5. considering the multiple fragile functional groups present in the taccalonolides and small quantity of natural taccalonolides available (less than 1 mg in some cases), a mild and efficient epoxidation method was needed. dimethyldioxirane (dmdo) can rapidly epoxidize alkenes under neutral and mild conditions, and it is also well suited for the synthesis of sensitive epoxides of enol esters and enol lactones, as are present in the taccalonolides. the reaction is highly efficient, generally furnishing the desired epoxides in almost quantitative yield. it is also very convenient, and in most cases pure products are obtained after evaporation of the solvent. due to the dramatic increases in potency conferred by the epoxidation of the c-22,23 double bond in 2 and 3 to generate 1 and 4, we applied this method to epoxidize a wide variety of other natural taccalonolides: e (7, 1.15 mg), n (6, 1.75 mg), r (8, 1.44 mg), t (9, 2.1 mg), z (10, 0.74 mg), aa (11, 1.0 mg), ab (12, 0.94 mg), ad (13, 1.28 mg), ai (5, 0.62 mg), and an (14, 0.65 mg) (see scheme 1). most of these natural taccalonolides are very rare with estimated content in the plant at ppm levels or less. only the epoxy was obtained as determined by the small coupling constant between h20/h22. we hypothesize that this is a of spatial strains of the , axial orientation of the 27-ch3. the antiproliferative potency of each c-22,c23 epoxy - taccalonolide was evaluated in hela cells and compared with the ic50 of the parent compound (table 1). in each case, c-22,23 epoxidation ed in an increase in potency with an over 200-fold improvement in potency observed for 5 of the 10 new epoxy - taccalonolides (table 1). remarkably, the c-22,23 epoxidation of 9 and 5 ed in the generation of the first ever compounds of this class with subnanomolar potencies of 0.45 and 0.88 nm, respectively. this makes 18 and 23 more potent than paclitaxel, which had an ic50 of 1.2 nm in this assay. the fold increase in potency achieved by c-22,23 expoxidation is shown in brackets in the last column. in addition to their potent antiproliferative effects, each epoxidized taccalonolide caused interphase microtubule bundling in hela cells. these effects are depicted in figure 2 for the two most potent taccalonolides, 18 and 23, and representative images of the cellular microtubule stabilizing effects of other, less potent epoxy taccalonolides are shown in the supporting information. in addition to their microtubule stabilizing effects in cells, both 18 and 23 enhanced the polymerization of purified porcine brain tubulin in turbidimetric assays (figure 3). similarly to other potent taccalonolides, including 1 and 4, they enhanced the extent of tubulin polymerization compared with vehicle controls without affecting the time required to initiate tubulin polymerization, a feature that makes this class of microtubule stabilizers distinct from other microtubule stabilizers that bind to the paclitaxel or laulimalide binding sites. microtubules were visualized by immunofluorescence using a -tubulin antibody after treatment of hela cells for 18 h with (a) vehicle (etoh), (b) 4 nm 18, or (c) 10 nm 23. purified porcine brain tubulin was incubated with 6 m 23, 0.7 m 18, or etoh vehicle, and microtubule polymerization was monitored turbidimetrically after shift to 37 c. despite the fact that 9 and 5 are rare natural products, both semisynthetic c-22,23 epoxidation products 23 and 18 antitumor studies could be performed with small quantities of material due to the exquisite potency of the taccalonolides in vivo. the ability of taccalonolides to covalently bind to microtubules likely contributes to their in vivo potency. the low doses of taccalonolides needed to observe effects in vivo allow them to be diluted in aqueous solvents, in this case less than 10% etoh in phosphate buffered saline (pbs). the antitumor efficacies of 18 and 23 were compared with nontreated tumors as a negative control and three doses of 15 mg / kg paclitaxel administered on days 0, 3, and 7 as a positive control for antitumor activity. individual doses of 0.25 mg / kg 18 were administered twice in the first week (days 0, 3) based on preliminary dose tolerance studies that showed a total dose of 0.5 mg / kg was acceptable. no additional drug was administered due to an average 10% body weight loss observed on day 7. despite the low total dose administered (0.5 mg / kg), tumor growth was completely inhibited through day 7 (figure 4). during days 714 , the mice gradually recovered to 4% body weight loss while significant antitumor effects were sustained for over a week after the final dose was administered (figure 4). by day 17 , the mice had fully recovered from drug - induced weight loss and some antitumor effects persisted. one lethality was encountered on day 21, which was 18 days after the final dose and after a full recovery of body weight loss; it is therefore unclear whether this was a drug - related toxicity. as has been noted for other taccalonolides, 18 has a narrow therapeutic window based on a concurrent study where two doses of 0.375 mg / kg administered on days 0 and 3 (0.75 total dose) ed in an lc40 with 2 of the 5 mice succumbing 811 days after the final dose. despite this narrow therapeutic window , the ability of a total dose of 0.5 mg / kg 18 to produce antitumor effects highlights the exceptional in vivo potency of the taccalonolides. taccalonolide 23 was administered at 0.75 mg / kg on days 0, 3, and 7 for a total dose of 2.25 mg / kg. only slight antitumor effects were observed, but dosing could not be increased due to a limited amount of material, which was fully expended on day 7. although 23 was only 2-fold less potent than 18 in vitro, no significant antitumor effects or weight loss were observed with 23 at a dose 3-fold higher than the dose of 18 that produced antitumor effects. although we can not rule out the possibility that 23 may have antitumor efficacy at higher concentrations, these highlight that in vitro potency can not be used as a sole predictor of in vivo efficacy, even when drugs of the same class are being compared. these are consistent with studies showing that 1 had excellent antitumor effects while 4 did not. in vivo efficacy of a cumulative total dose of 2.25 mg / kg 23 or 0.5 mg / kg 18 in an mda - mb-231 xenograft model of triple negative breast cancer compared with a cumulative total dose of 45 mg / kg paclitaxel. the role of this carbonyl group in the microtubule stabilizing activity of the taccalonolides is unknown. reduction of this carbonyl group with nabh4 ed in two new semisynthetic products, 25 and 26, that were isolated with respective 2% and 9% yields after hplc purification (scheme 2). due to the spatial strain of the orientation of both 18-ch3 and 7-oh, we hypothesize that the hydride attached to the carbonyl from the face ing in the 6 reduction products of 25 and 26. compound 25 was obtained as white powder, and a molecular formula of c34h46o13 was deduced from the hrms, 663.3021 (calcd for c34h47o13 663.3011). the h nmr showed signals only for three acetyl methyl groups, suggesting the loss of one acetyl group. the chemical shift of h-15 at 4.39 (t, j = 8.7 hz), ca. 1.1 ppm higher than that of taccalonolide a, indicated that the acetyl group at 15-oh was lost. this is consistent with previous studies showing that the c-15 acetoxy can easily be hydrolyzed. the c-6 carbonyl signal was also lost, and instead, signals for a hydroxymethine at h 3.80 (br, h-6) and c 73.5 (c-6) were observed, indicating the successful reduction of the c-6 carbonyl group. the orientation of the 6-oh was determined to be (equatorial) due to the small coupling constant of h-6. thus, 25 is a product of the reduction of the c-6 carbonyl and the hydrolysis of the c-15 acetoxyl group. the molecular formula of c36h48o14 was determined by hrms, 705.3137 (calcd for c36h49o14 705.3168), corresponding to the reduction of a carbonyl group. resonance at 4.37, suggested the hydrolysis of this acetyl group to give 15-oh. interestingly, this acetyl group appears to shift to the newly generated c-6 hydroxyl group based on the h 5.07 (br, h-6), c 76.7 (c-6), and cosy correlations between h-6/h-5 (2.05, m), h-6/h-7 (3.79, dd, j = 9.5, 2.5 hz), and h-7/h-8 (1.94, m). the configuration of 6-oac was determined to be (equatorial) due to the small coupling constant of h-6. we hypothesize that the migration of the acetyl group from 15-oh to 6-oh might have occurred through two steps (see scheme 3). the 15-acetyl group could have first migrated to 7-oh since they are very close and the 15-acetoxy is prone to loss of the acetyl group as demonstrated previously. then the newly formed alkoxide from the reduction of the 6-ketone could have attached to the carbonyl carbon of the 7-oac group to yield 26, the 6-oac product. both 25 and 26 were found to have no antiproliferative effects at concentrations up to 50 m, suggesting the importance of the c-6 ketone for activity. similarly to the observed in table 1, c-22,23 epoxidation of 26 ed in an over 300-fold improvement of potency to generate an active taccalonolide, 27, with an ic50 of 163 10 nm. the newly isolated taccalonolide 5 and the known taccalonolide 9 showed relatively potent antiproliferative activities with ic50 values of 47 and 335 nm, respectively, which are 181- and 39-fold more potent than 6 and 8. the only difference between taccalonolides 5/6 and 9/8 is that both 5 and 9 have an isovaleryloxy group at c-1, while 6 and 8 have an acetyloxy group at c-1. this supports previous assertions that a bulky substitution at c-1 is preferred for the antiproliferative activity of the taccalonolides. epoxidation of the c-22,23 double bond significantly increased the potency of every taccalonolide analyzed. taking into account 1 and 4 (the epoxy products of 2 and 3, respectively), epoxidation of the c-22,23 double bond ed in an over 200-fold increase in potency for 7 of the 12 natural taccalonolides tested (table 1). although these data show that a c-22,23 epoxide is optimal for the potency of a wide range of taccalonolides, further information can be gleaned from the relative effect that this epoxidation has on activity. two taccalonolides, 10 and 11, showed relatively modest 7- and 2-fold increases in potency, respectively, after epoxidation. however, it is important to note that these two taccalonolides were relatively potent before epoxidation (120 and 32 nm, respectively) and that their epoxidized forms are some of the most potent taccalonolides identified to date (table 1). therefore, it appears that there may be a maximum potency that can be conferred to some taccalonolides by epoxidation. the notable outliers to this are the epoxidized forms of 9 and 5, which each contain a bulky isovaleryloxy group at c-1 and are the first taccalonolides to show subnanomolar potency. together, these suggest that the combination of a c-22,23 epoxide with a bulky c-1 modification is optimal for taccalonolide potency. taccalonolides 13 and 14 also showed only modest increases of 24-fold in potency after c-22,23 epoxidation. these compounds differ from those previously mentioned in that even the epoxidized forms, 22 and 24, have only moderate potencies, between 685820 nm. therefore, although epoxidation increased the potency of these taccalonolides, the ing activities remained modest. we hypothesize that these nonoptimal substituents on c-6 and c-7 limit the potency of 13 even when epoxidized at c-22,23. a similar limit in potency was conferred by the hydrolysis of the c-1 group in 14. both c-6 reductive products of the major metabolite taccalonolide a, 25 and 26, exhibited decreased potency, further demonstrating the importance of the c-6 ketone for activity. however, the finding that c-22,23 epoxidation of 26 leads to an over 300-fold improvement in potency demonstrates that this epoxidation can dramatically improve the activity even of taccalonolides that otherwise have no detectable antiproliferative effects. together, these data provide more extensive sar for the taccalonolides, including the critical importance of c-22,23, c-1, and c-6 modifications as well as the interplay between these substituents. each of the 11 taccalonolides epoxidized at c-22,23 in this study exhibited increased potency compared with their precursors, the majority showing over 200-fold improvement in activity. two of these products, 18 and 23, are the first taccalonolides identified with subnanomolar potency. these two epoxidized taccalonolides have potent microtubule stabilizing activities in cells and with purified tubulin, but only 18 demonstrated antitumor efficacy at the dose and schedule tested. these demonstrate that a c-22,23 epoxy combined with a bulky c-1 isovaleryloxy group facilitates optimal potency for the taccalonolide class of microtubule stabilizers and further enhances our understanding of the structure while both c-22,23 epoxidated and nonepoxidated taccalonolides cause microtubule stabilization in cells, the recent finding that some epoxy taccalonolides can covalently bind to microtubules leads to a hypothesis that the c-22,23 epoxide may facilitate their irreversible binding, which would be consistent with the increased potency afforded by this modification. additional studies nmr spectra were acquired on bruker avance 500, 600, or 700 mhz instruments equipped with cryoprobes using cdcl3 as solvent. all spectra were measured and reported in ppm using trimethylsilane as an internal standard. the hrms data were obtained on an aglient technologies 6224 toflc / ms mass spectrometer. lc / ms was performed with a waters alliance 2695 hplc module, 996 photodiode array detector, and micromass quattro triple quadrupole mass spectrometer equipped with esi under the positive mode. tlc was performed on aluminum sheets (silica gel 60 f254, merck kgaa, germany). spots were visualized by spraying with 20% sulfuric acid in ethanol followed by heating. taccalonolides e, n, r, t, z, aa, ab, ad, and an were obtained previously. a taccalonolide b enriched fraction was further purified by reversed phase hplc (phenomenex luna, 5 m c18 250 21.2 mm column) eluting with a gradient of 40100% acetonitrile in h2o in 50 min to yield highly pure 3 and the minor product, taccalonolide ai. white powder. hrms (m / z) calcd for c35h49o11 645.3269, found 645.3268; h nmr (500 mhz, cdcl3) 5.23 (d, j = 2.6 hz, 1h, oh-15), 5.02 (br, 1h, h-22), 4.99 (t, j = 2.8 hz, 1h, h-12), 4.72 (s, 1h, oh-25), 4.59 (d, j = 5.2 hz, 1h, h-1), 4.45 (br, 1h, oh-7), 4.38 (ddd, j = 9.5, 8.3, 2.5 hz, 1h, h-15), 4.01 (d, j = 10.3 hz, 1h, h-7), 3.55 (t, j = 4.0 hz, 1h, h-2), 3.40 (br, 1h, h-3), 2.70 (dd, j = 11.3, 4.5 hz, 1h, h-5), 2.39 (dd, j = 13.1, 10.9 hz, 1h, h-16), 2.26 (dd, j = 16.1, 4.5 hz, 1h, h-4a), 2.19 (m, 1h, h-20), 2.18 (m, 2h, h-2), 2.15 (m, 1h, h-9), 2.14 (m, 1h, h-3), 2.12 (m, 1h, h-4b), 2.08 (s, 3h, 12ococh3), 2.07 (m, 1h, h-14), 1.98 (dd, j = 13.2, 10.1 hz, 1h, h-17), 1.71 (m, 1h, h-8), 1.69 (m, 2h, h-11), 1.67 (s, 3h, h-28), 1.36 (s, 3h, h-27), 1.01 (d, j = 7.0 hz, 3h, h-4), 1.00 (d, j = 6.0 hz, 3h, h-5), 0.95 (d, j = 7.0 hz, 3h, h-21), 0.82 (s, 3h, h-19), 0.76 (s, 3h, h-18). c nmr (125 mhz, cdcl3) 209.1 (c-6), 175.4 (c-26), 171.8 (c-1), 169.3 (12-ococh3), 154.9 (c-23), 110.4 (c-22), 78.9 (c-25), 77.5 (c-7), 73.5 (c-12), 71.8 (c-15), 70.4 (c-1), 57.3 (c-14), 52.5 (c-3), 50.8 (c-24), 49.9 (c-16), 49.3 (c-2), 47.7 (c-17), 43.8 (c-13), 43.4 (c-5), 42.6 (c-2), 42.2 (c-8), 41.2 (c-10), 36.1 (c-9), 30.8 (c-20), 25.1 (c-27), 25.4 (c-3), 24.7 (c-11), 21.9 (c-4,5), 21.7 (c-28), 20.9 (c-4), 20.7 (12-ococh3), 19.3 (c-21), 12.9 (c-18), 12.3 (c-19). dimethyldioxirane was prepared by reaction of oxone with acetone, and the concentration of dimethyldioxirane was determined by uv. 13 mol ) was dissolved in 500 l of ch2cl2 and cooled to 20 c. dmdo (23 equiv) was added, and the mixture was stirred at room temperature until the taccalonolide was completely epoxidized (14 h). the epoxides were obtained after removal of the solvents and reagent with no further purification required. white powder. hrms (m / z) calcd for c34h45o13 661.2854, found 661.2851; h nmr (500 mhz, cdcl3) 5.47 (t, j = 9.1 hz, 1h, h-15), 4.91 (d, j = 3.0 hz, 1h, h-12), 4.60 (d, j = 5.3 hz, 1h, h-1), 3.86 (dd, j = 10.1, 3.7 hz, 1h, h-7), 3.83 (br, 7-oh), 3.51 (t, j = 4.7 hz, 1h, h-2), 3.39 (br, 1h, h-3), 3.30 (s, 1h, h-22), 2.67 (m, 2h, h-5, 25-oh), 2.32 (dd, j = 10.9, 8.6 hz, 1h, h-14), 2.262.03 (m, 6h), 2.12 (s, 3h, 1-ococh3), 2.10 (s, 3h, 12-ococh3), 2.01 (s, 3h, 15-ococh3), 1.76 (s, 3h, h-28), 1.751.60 (m, 4h), 1.35 (s, 3h, h-27), 1.08 (d, j = 7.4 hz, 3h, h-21), 0.75 (s, 3h, h-19), 0.70 (s, 3h, h-18). white powder. hrms (m / z) calcd for c32h43o12 619.2749, found 619.2757; h nmr (500 mhz, cdcl3) 5.25 (d, j = 3.1 hz, 1h, h-12), 5.08 (br, 1h, oh), 4.88 (d, j = 3.0 hz, 1h, oh), 4.59 (d, j = 5.4 hz, 1h, h-1), 4.44 (br, 1h, oh), 4.30 (t, j = 10.0 hz, 1h, h-15), 4.01 (d, j = 10.4 hz, 1h, h-7), 3.53 (t, j = 4.3 hz, 1h, h-2), 3.41 (br, 1h, h-3), 3.28 (s, 1h, h-22), 2.71 (dd, j = 11.7, 4.7 hz, 1h, h-5), 2.312.03 (m, 6h), 2.10 (s, 3h, 1-ococh3), 2.09 (s, 3h, 12-ococh3), 1.76 (s, 3h, h-28), 1.751.67 (m, 4h), 1.37 (s, 3h, h-27), 1.07 (d, j = 7.3 hz, 3h, h-21), 0.74 (s, 3h, h-19), 0.71 (s, 3h, h-18). white powder. hrms (m / z) calcd for c36h47o15 719.2910, found 719.2907; h nmr (500 mhz, cdcl3) 5.55 (d, j = 10.8 hz, 1h, h-7), 5.50 (t, j = 8.6 hz, 1h, h-15), 4.88 (br, 1h, h-12), 4.81 (d, j = 5.1 hz, 1h, h-1), 3.70 (t, j = 4.5 hz, 1h, h-2), 3.58 (d, j = 3.8 hz, 1h, h-3), 3.31 (s, 1h, 5-oh), 3.29 (s, 1h, h-22), 2.82 (td, j = 10.8, 7.1 hz, 1h, h-9), 2.70 (s, 1h, 25-oh), 2.542.47 (m, 2h, h-4a,14), 2.261.91 (m, 6h), 2.16 (s, 3h, 1-ococh3), 2.14 (s, 3h, 7-ococh3), 2.12 (s, 3h, 12-ococh3), 1.97 (s, 3h, 15-ococh3), 1.73 (s, 3h, h-28), 1.67 (m, 3h, h2 - 11, h-20), 1.33 (s, 3h, h-27), 0.80 (d, j = 7.4 hz, 3h, h-21), 0.78 (s, 3h, h-19), 0.70 (s, 3h, h-18). white powder. hrms (m / z) calcd for c39h53o15 761.3379, found 761.3362; h nmr (600 mhz, cdcl3) 5.55 (d, j = 10.7 hz, 1h, h-7), 5.50 (t, j = 8.7 hz, 1h, h-15), 4.88 (t, j = 3.2 hz, 1h, h-12), 4.79 (d, j = 5.1 hz, 1h, h-1), 3.72 (t, j = 4.4 hz, 1h, h-2), 3.57 (br, 1h, h-3), 3.30 (br, 1h, 5-oh), 3.29 (br, 1h, h-22), 2.82 (dt, j = 11.6, 8.5 hz, 1h, h-9), 2.72 (s, 1h, 25-oh), 2.50 (m, 2h, h-4a,14), 2.252.16 (m, 5h, h4b, h-17, h2 - 2, h-3), 2.16 (s, 3h, 7-ococh3), 2.13 (s, 3h, 12-ococh3), 1.97 (s, 3h, 15-ococh3), 1.95 (m, 1h, h-8), 2.02 (dd, j = 13.9, 9.2 hz, 1h, h-16), 1.73 (s, 3h, h-28), 1.67 (m, 3h, h2 - 11, h-20), 1.32 (s, 3h, h-27), 1.07 (d, j = 7.3 hz, 3h, h-21), 1.01 (t, j = 6.6 hz, 6h, h-4,5), 0.77 (s, 3h, h-19), 0.70 (s, 3h, h-18). c nmr (150 mhz, cdcl3) 201.5 (c-6), 177.9 (c-26), 172.1 (1-ococh3), 172.3 (15-ococh3), 171.4 (7-ococh3), 169.7 (12-ococh3), 92.8 (c-23), 79.9 (c-5), 79.6 (c-25), 76.4 (c-7), 74.5 (c-12), 72.5 (c-1), 72.0 (c-15), 66.2 (c-22), 55.0 (c-3), 54.4 (c-14), 50.8 (c-2), 48.5 (c-16), 46.9 (c-24), 45.1 (c-17), 44.9 (c-13), 44.7 (c-10), 43.4 (c-2), 34.4 (c-9), 32.0 (c-20), 27.3 (c-4), 26.6 (c-3), 26.4 (c-11), 23.9 (c-28), 23.4 (15-ococh3), 22.9 (c-4,5), 21.9 (7,12-ococh3), 20.0 (c-27), 18.9 (c-21), 15.1 (c-18), 13.9 (c-19). white powder. hrms (m / z) calcd for c36h47o16 735.2859, found 735.2867; h nmr (500 mhz, cdcl3) 5.53 (t, j = 8.9 hz, 1h, h-15), 5.19 (brd, j = 12.2 hz, 1h, h-11), 5.16 (br, 1h, h-12), 4.85 (d, j = 5.3 hz, 1h, h-1), 4.71 (dd, j = 10.3, 5.2 hz, 1h, h-7), 3.74 (t, j = 4.7 hz, 1h, h-2), 3.64 (br, 1h, 5-oh), 3.61 (br, 1h, h-3), 3.47 (d, j = 4.9 hz, 1h, 7-oh), 3.27 (s, 1h, h-22), 3.16 (t, j = 11.5 hz, 1h, h-9), 2.66 (s, 1h, 25-oh), 2.57 (d, j = 16.5 hz, 1h, h-4a), 2.50 (t, j = 10.0 hz, 1h, h-14), 2.23 (d, j = 16.5 hz, 1h, h-4b), 2.17 (s, 3h, 1-ococh3), 2.16 (s, 3h, 12-ococh3), 2.152.03 (m, 2h, h-16,17), 2.09 (s, 3h,11-ococh3), 1.98 (s, 3h, 15-ococh3), 1.76 (s, 3h, h-28), 1.58 (m, 1h, h-20), 1.34 (s, 3h, h-27), 1.01 (d, j = 7.4 hz, 3h, h-21), 0.88 (s, 3h, h-19), 0.72 (s, 3h, h-18). white powder. hrms (m / z) calcd for c38h49o17 777.2964, found 777.2947; h nmr (500 mhz, cdcl3) 5.70 (d, j = 10.9 hz, 1h, h-7), 5.53 (t, j = 8.3 hz, 1h, h-15), 5.31 (br, 1h, 5-oh), 5.19 (d, j = 12.6 hz, 1h, h-11), 5.18 (br, 1h, h-12), 4.90 (d, j = 5.2 hz, 1h, h-1), 3.72 (t, j = 4.8 hz, 1h, h-2), 3.59 (br, 1h, h-3), 3.28 (m, 2h, h-9,22), 2.69 (s, 1h, 25-oh), 2.60 (t, j = 10.2 hz, 1h, h-14), 2.56 (d, j = 17.1 hz, 1h, h-4a), 2.152.00 (m, 3h, h-4b, 8,16), 2.20 (s, 3h, 1-ococh3), 2.17 (s, 3h, 7-ococh3), 2.16 (s, 3h, 12-ococh3), 1.99 (s, 6h, 11,15-ococh3), 1.75 (s, 3h, h-28), 1.62 (m, 2h, h-17,20), 1.32 (s, 3h, h-27), 1.09 (d, j = 7.3 hz, 1h), 1.02 (d, j = 7.3 hz, 3h, h-21), 0.93 (s, 3h, h-19), 0.71 (s, 3h, h-18). white powder. hrms (m / z) calcd for c34h45o15 693.2753, found 693.2771; h nmr (500 mhz, cdcl3) 5.30 (s, 1h, 25-oh), 5.22 (dd, j = 12.0, 2.5 hz, 1h, h-11), 5.13 (d, j = 2.4 hz, 1h, h-12), 4.96 (br, 2h, 15,25-oh), 4.91 (dd, j = 10.6, 4.5 hz, 1h, h-7), 4.81 (d, j = 5.4 hz, 1h, h-1), 4.38 (td, j = 9.1, 3.7 hz, 1h, h-15), 4.03 (d, j = 4.5 hz, 1h, 7-oh), 3.75 (t, j = 4.5 hz, 1h, h-2), 3.68 (s, 1h, 5-oh), 3.62 (br, 1h, h-3), 3.25 (s, 1h, h-22), 3.15 (t, j = 11.6 hz, 1h, h-9), 2.54 (dd, j = 16.6, 2.1 hz, 1h, h-4a), 2.27 (d, j = 16.6, hz, 1h, h-4b), 2.21 (m, 1h, h-14), 2.17 (s, 3h, 1-ococh3), 2.15 (s, 3h, 12-ococh3), 2.09 (m, 2h, h-16,17), 1.98 (s, 3h, 11-ococh3), 1.77 (s, 3h, h-28), 1.61 (m, 2h, h-8,20), 1.36 (s, 3h, h-27), 0.99 (d, j = 7.4 hz, 3h, h-21), 0.85 (s, 3h, h-19), 0.76 (s, 3h, h-18). white powder. hrms (m / z) calcd for c36h45o15 717.2753, found 717.2738; h nmr (500 mhz, cdcl3) 6.19 (s, 1h, 6-oh), 5.64 (t, j = 8.5 hz, 1h, h-15), 5.43 (dd, j = 11.3, 2.3 hz, 1h, h-11), 5.26 (d, j = 2.3 hz, 1h, h-12), 4.90 (d, j = 5.5 hz, 1h, h-1), 3.55 (t, j = 4.2 hz, 1h, h-2), 3.41 (t, j = 3.7 hz, 1h, h-3), 3.38 (d, j = 19.7 hz, 1h, h-4a), 3.28 (br, 1h, h-22), 2.83 (t, j = 12.3 hz, 1h, h-11), 2.74 (s, 1h, 25-oh), 2.62 (m, 3h, h-4b,8,14), 2.44 (dd, j = 11.4, 8.0 hz, 1h, h-16), 2.18 (s, 3h, 15-ococh3), 2.11 (s, 3h, 1-ococh3), 2.10 (s, 3h, 11-ococh3), 2.00 (s, 3h, 12-ococh3), 1.75 (m, 2h, h-17,20), 1.71 (s, 3h, h-28), 1.31 (s, 3h, h-27), 1.21 (s, 3h, h-18), 1.07 (d, j = 6.8 hz, 3h, h-21), 0.92 (s, 3h, h-19). white powder. hrms (m / z) calcd for c35h49o12 661.3219, found 661.3211; h nmr (500 mhz, cdcl3) 5.26 (d, j = 3.0 hz, 1h, 15-oh), 5.08 (s, 1h, 25-oh), 4.90 (t, j = 2.7 hz, 1h, h-12), 4.57 (d, j = 5.3 hz, 1h, h-1), 4.44 (d, j = 3.1 hz, 1h, 7-oh), 4.29 (t, j = 9.5 hz, 1h, h-15), 4.01 (d, j = 10.3 hz, 1h, h-7), 3.55 (t, j = 4.4 hz, 1h, h-2), 3.40 (d, j = 3.2 hz, 1h, h-3), 3.27 (s, 1h, h-22), 2.69 (dd, j = 11.4, 4.8 hz, 1h, h-5), 2.27 (d, j = 16.7, 5.0 hz, 1h, h-4a), 2.242.02 (m, 7h, h-4b,9,14,16,3, h22), 2.10 (s, 3h, 12-ococh3), 1.67 (s, 3h, h2 - 11, h-20), 1.76 (s, 3h, h-28), 1.37 (s, 3h, h-27), 1.06 (d, j = 7.4 hz, 3h, h-21), 1.02 (dd, j = 6.2, 3.6 hz, 6h, h-4,5), 0.75 (s, 3h, h-19), 0.71 (s, 3h, h-18). white powder. hrms (m / z) calcd for c30h41o11 577.2643, found 577.2657; h nmr (500 mhz, cdcl3) 5.18 (d, j = 3.1 hz, 1h, 15-oh), 5.08 (s, 1h, h-12), 4.94 (br, 1h, 25-oh), 4.30 (t, j = 9.9 hz, 1h, h-15), 4.35 (br, 1h, 7-oh), 3.98 (d, 1h, j = 10.3 hz, 1h, h-7), 3.71 (dd, j = 8.4, 5.7 hz, 1h, h-1), 3.55 (br, 1h, h-3), 3.42 (t, j = 5.1 hz, 1h, h-2), 3.28 (br, 1h, h-22), 2.51 (dd, j = 11.1, 5.1 hz, 1h, h-5), 2.282.11 (m, 4h, h2 - 4, h-9,16), 2.10 (s, 3h, 12-ococh3), 2.082.02 (m, 2h, h-14,17), 1.76 (s, 3h, h-28), 1.701.58 (m, 2h, h-8,11), 1.37 (s, 3h, h-17), 1.08 (d, j = 7.2 hz, 1h, h-21), 0.72 (s, 3h, h-19), 0.63 (s, 3h, h-18). compound 2 (10.2 mg) was dissolved in 2 ml of meoh and cooled in a sodium chloride ice bath. the ch2cl2 soluble material was subjected to hplc separation to yield compounds 25 (0.15 mg) and 26 (0.93 mg). white powder. hrms (m / z) calcd for c34h47o13 663.3011, found 663.3021; h nmr (600 mhz, cdcl3) 5.40 (d, j = 2.9 hz, 1h, 15-oh), 5.35 (dd, j = 11.7, 2.8 hz, 1h, h-11), 5.23 (d, j = 2.8 hz, 1h, h-12), 5.01 (s, 1h, h-22), 4.64 (s, 25-oh), 4.56 (d, j = 5.4 hz, 1h, h-1), 4.38 (t, j = 8.9 hz, 1h, h-15), 3.80 (s, 1h, h-6), 3.51 (m, 1h, h-7), 3.47 (m, 1h, h-2), 3.39 (s, 1h, h-3), 2.39 (dd, j = 13.4, 10.6 hz, 1h, h-16), 2.25 (t, j = 13.8 hz, 1h, h-4a), 2.15 (m, 2h, h-9,20), 2.11 (s, 3h, 1-ococh3), 2.07 (s, 3h, 12-ococh3), 1.95 (s, 3h, 11-ococh3), 2.00 (m, 1h, h-8), 1.97 (m, 1h, h-14), 1.94 (m, 1h, h-4b), 1.70 (m, 2h, h-5,17), 1.66 (s, 3h, h-28), 1.34 (s, 3h, h-27), 1.24 (s, 1h), 1.02 (s, 3h, h-18), 0.99 (s, 3h, h-19), 0.91 (d, j = 7.1 hz, 3h, h-21). c nmr (150 mhz, cdcl3) 176.2 (c-26), 171.3 (11-ococh3), 170.2 (1-ococh3), 169.8 (12-ococh3), 154.8 (c-23), 111.2 (c-22), 78.9 (c-25), 74.2 (h-11), 74.1 (2xc, c-1,7), 73.5 (c-6), 72.0 (c-15), 71.0 (c-12), 56.3 (c-14), 50.9 (c-24), 48.2 (c-17), 44.6 (c-13), 40.5 (c-10), 37.5 (c-9), 35.3 (c-8), 33.4 (c-5), 31.4 (c-20), 26.0 (c-4), 25.6 (c-27), 22.0 (c-28), 21.7 (11-ococh3), 21.3 (12-ococh3), 21.0 (1-ococh3), 20.5 (c-21), 14.6 (c-18), 13.7 (c-19). white powder. hrms (m / z) calcd for c36h49o14 705.3168, found 705.3137; h nmr (500 mhz, cdcl3) 5.47 (s, 1h, 15-oh), 5.36 (dd, j = 11.7, 2.6 hz, 1h, h-11), 5.23 (d, j = 2.6 hz, 1h, h-12), 5.06 (t, j = 2.7 hz, 1h, h-6), 4.98 (d, j = 1.1 hz, 1h, h-22), 4.74 (br, 1h, oh), 4.64 (d, j = 5.5 hz, 1h, h-1), 4.36 (t, j = 9.2 hz, 1h, h-15), 3.78 (d, j = 7.0 hz, 1h, h-7), 3.59 (s, 1h, oh), 3.46 (dd, j = 5.1, 3.9 hz, 1h, h-2), 3.34 (d, j = 3.3 hz, 1h, h-3), 2.37 (dd, j = 13.3, 10.6 hz, 1h, h-16), 2.22 (m, 1h, h-9), 2.21 (s, 3h, 6-ococh3), 2.17 (m, 1h, h-20), 2.12 (s, 3h, 1-ococh3), 2.06 (s, 3h, 12-ococh3), 2.05 (m, 1h, h-5), 1.97 (s, 3h, 11-ococh3), 1.981.92 (m, 4h, h-8,14, h2 - 4), 1.80 (dd, j = 13.4, 9.8 hz, 1h, h-17), 1.65 (s, 3h, h-28), 1.32 (s, 1h, h-27), 1.03 (s, 1h, h-18), 1.01 (s, 1h, h-19), 0.90 (d, j = 7.0 hz, 3h, h-21). c nmr (125 mhz, cdcl3) 175.5 (c-26), 173.7 (6-ococh3), 170.8 (11-ococh3), 169.6 (1-ococh3), 169.4 (12-ococh3), 154.5 (c-23), 110.4 (c-22), 78.9 (c-25), 76.2 (c-6), 74.0 (c-7), 73.6 (c-12), 73.4 (c-1), 71.2 (c-15), 70.5 (c-11), 56.1 (c-14), 52.0 (c-3), 51.2 (c-16), 50.7 (c-24), 49.9 (c-2), 47.6 (c-17), 44.2 (c-13), 39.9 (c-9), 37.0 (c-10), 34.8 (c-8), 31.7 (c-5), 30.8 (c-20), 25.2 (2xc, c-4,27), 21.6 (c-28), 21.0 (11-ococh3), 20.7 (1,6-ococh3), 20.4 (12-ococh3), 20.1 (c-21), 13.9 (c-18), 13.3 (c-19). white powder. hrms (m / z) calcd for c36h49o15 721.3066, found 721.3043; h nmr (600 mhz, cdcl3) 5.53 (d, j = 2.8 hz, 1h, 15-oh), 5.32 (dd, j = 11.8, 2.9 hz, 1h, h-11), 5.15 (d, j = 2.9 hz, 1h, h-12), 5.11 (s, 1h, oh), 5.05 (t, j = 2.8 hz, 1h, h-6), 4.63 (d, j = 5.6 hz, 1h, h-1), 4.27 (brt, j = 7.2 hz, 1h, h-15), 3.79 (brd, j = 9.5 hz, 1h, h-7), 3.53 (s, 1h, oh), 3.46 (m, j = 4.7 hz, 1h, h-2), 3.33 (br, 1h, h-3), 3.23 (s, 1h, h-22), 2.21 (s, 3h, 6-ococh3), 2.261.87 (m, 9h, h2 - 4, h-5,8,9,14,16,17,20), 2.12 (s, 3h, 1-ococh3), 2.08 (s, 3h, 12-ococh3), 1.96 (s, 3h, 11-ococh3), 1.73 (s, 3h, h-28), 1.32 (s, 3h, h-27), 1.02 (s, 3h, h-18), 1.01 (d, j = 7.4 hz, 3h, h-21), 0.90 (s, 3h, h-19). the antiproliferative activities and ic50 values of the taccalonolides were determined using the sulforhodamine b assay in hela cells as previously described. the data are from an average of three experiments, each performed in triplicate, with standard deviation. microtubules in interphase hela cells were visualized by immunofluorescence using a -tubulin antibody (sigma no . images were acquired 18 h after indicated drug or vehicle treatment using a nikon eclipse 80i fluorescence microscope and nis elements ar 3.0 software . the ability of the taccalonolides 23 and 18 to enhance tubulin polymerization was determined as previously described . briefly, 1 l of a 100 stock solution of each compound in etoh was added to a final volume of 100 l containing 2 mg / ml purified porcine brain tubulin ( cytoskeleton) in gpem buffer (80 mm na - pipes, ph 6.9, 1 mm egta, 1 mm mgcl2, 1 mm gtp, and 10% glycerol), and microtubule formation was monitored turbidimetrically at od340 on a spectramax 96-well plate reader. the antitumor efficacy of the two most potent taccalonolides, 23 and 18, was evaluated in a mda - mb-231 triple negative breast cancer xenograft model. tumor fragments were bilaterally implanted in female nude (nu / nu) mice. mice were randomly placed into separate treatment groups (n = 5), and dosing was initiated when the average tumor size was 134 mm. taccalonolides 23 and 18 were solubilized in 100% etoh at 1 and 1.25 mg / ml, respectively, while paclitaxel stocks were solubilized in 50% cremophor/50% etoh at 15 mg / ml. drug stocks were diluted a minimum of 1:10 in 200 l of phosphate buffered saline immediately prior to intraperitoneal injection. dose and schedule taccalonolide 23 was administered at a concentration of 0.7 mg / kg on days 0, 3, and 7 after which dosing was halted due to expenditure of all available material. taccalonolide 18 was administered at a concentration of 0.25 mg / kg on days 0 and 3, after which dosing was halted due to an average 10% weight loss. paclitaxel was administered at 15 mg / kg on days 0, 3, and 7. during the period of drug administration, mice were monitored daily, and weight and tumor measurements were taken 23 times weekly. control treated mice were sacrificed on day 17 due to large tumor size; all other mice were sacrificed on day 21. the mice were purchased from harlan laboratories, housed in an aalac - approved facility under fully licensed veterinary care, and provided water and food ad libitum.

the taccalonolides are microtubule stabilizers isolated from plants of the genus tacca. taccalonolide af is 231 times more potent than the major metabolite taccalonolide a and differs only by the oxidation of the c-22,23 double bond in a to an epoxy group in af. in the current study, 10 other rare natural taccalonolides were epoxidized and in each case epoxidation improved potency. the epoxidation products of taccalonolide t and ai were the most potent, with ic50 values of 0.43 and 0.88 nm, respectively. these potent taccalonolides retained microtubule stabilizing effects, and t - epoxide demonstrated antitumor effects in a xenograft model of breast cancer. additional reactions demonstrated that reduction of the c-6 ketone ed in an inactive taccalonolide and that c-22,23 epoxidation restored its activity. these studies confirm the value of c-22,23 epoxidation as an effective strategy for increasing the potency of a wide range of structurally diverse taccalonolide microtubule stabilizers.

here, we report the synthesis and characterization of size - controllable and stimuli - responsive dna nanohydrogels as effective targeted gene delivery vectors. dna nanohydrogels were created through a self - assembly process using three kinds of building units, respectively termed y - shaped monomer a with three sticky ends (yma), y - shaped monomer b with one sticky end (ymb), and dna linker (lk) with two sticky ends. hybridization at the sticky ends of monomers and lk leads to nanohydrogel formation. dna nanohydrogels are size - controllable by varying the ratio of yma to ymb. by incorporating different functional elements, such as aptamers, disulfide linkages, and therapeutic genes into different building units, the synthesized aptamer - based nanohydrogels (y - gel - apt) can be used for targeted and stimuli - responsive gene therapy. y - gel - apt strongly inhibited cell proliferation and migration in target a549 cells, but not in control cells. by taking advantage of facile modular design and assembly, efficient cellular uptake, and superior biocompatibility, this y - gel - apt holds great promise as a candidate for targeted gene or drug delivery and cancer therapy.

understanding a spoken message in the presence of noise or competing speech is a common listening problem for many older adults. the recognition process in the presence of competing signals is naturally complicated since it includes the encoding of each input signal at the peripheral level, followed by processing of the central auditory and cognitive systems. when age - related internal changes occur in peripheral, central, and general cognitive functions, any one of these changes can be sufficient to affect the understanding of competing speech signals, but older adults often experience changes at multiple levels of processing. when the target and competing voices are presented together, young adults are able to use differences in the fundamental frequency (f0) of competing speech signals in order to better identify the target message against the interfering message. however, this important cue may not be ideally processed for older listeners, revealed by numerous studies that reported the age - related disadvantage in the processing of f0 for the intelligibility of competing signals. thus, it is important to determine any negative impact of aging on the use of f0 cues between two competing speech signals, and various theoretical models which describe possible reasons of the age - related deficits above. this article addresses three issues, 1 ) theoretical hypotheses about age - related deficits on general speech communication, 2 ) previous findings on the perceptual benefits of f0 for competing speech signals and the age - related deficits on these benefits, and 3 ) theoretical models on the processing of f0 in order to compare which better explains the age - related disadvantage in the processing of f0. a working group of the committee on hearing and bioacoustics and biomechanics of the national research council reviewed the issues of the speech communication problems in older adults. as a possible explanation of age - related deficits on speech communication, the sensori - neural hearing loss and cochlear pathology of older listeners can in difficulties for the peripheral encoding of input sounds, and the age - related decline in central - auditory and general cognitive functions can also adversely influence processing of deteriorated input signals.1 ) when the age - related peripheral deficit in hearing sensitivity, often referred to as presbycusis, is the sole deficit, the listener's understanding of the target message is primarily affected by the inaudibility of the target speech arising from the presence of cochlear pathology.2 - 6 ) because this age - related hearing loss rarely in total deafness, most of older adults with presbycusis can still hear speech, but often have difficulty understanding speech. given the normal involvement of processing at peripheral, central - auditory, and cognitive levels in speech understanding, three hypotheses (i.e., auditory peripheral hypothesis, central - auditory hypothesis, and general cognitive hypothesis) have been used to explain the speech - understanding difficulties of the elderly adults with impaired hearing.1,7 ) age - related structural and functional changes in the auditory periphery have been assumed to directly affect the audibility and the processing of speech sounds in the peripheral hypothesis. the age - related peripheral hearing loss often starts in the high frequencies, with a progressive deterioration at high frequencies that is faster than at low frequencies. due to the high - frequency hearing loss, some high - frequency, low - intensity speech sounds, usually consonantal sounds such as " t, f, s, th ", can become inaudible even when a single talker speaks at typical conversational levels (60 - 65 db spl) in quiet conditions.8 ) many previous studies have consistently found that older individuals' speech - understanding performance is well - explained by the audibility of speech, estimated from hearing thresholds, when a single talker speaks in quiet or in a steady - state noise.3,7 - 11 ) when a target speech signal is presented with a noise that fluctuates like speech or with competing speech, individual differences in the speech - understanding performance of older individuals are not predicted by audibility alone. rather, age - related deficits often emerge as primary or strong secondary predictors.11,12 ) the role of age - related deficits in central - auditory and general cognitive process has also been confirmed in several studies.2,11 - 16 ) once the reduced speech audibility of the target message has been alleviated by clinical amplification or laboratory spectral shaping, other factors associated with central and cognitive deficits emerge, especially while listening to speech with competing speech in the . when the target and interfering messages are presented concurrently, spectral and temporal features of both competing speech signals are encoded and contrasted in order to segregate the target from the competing source. for the procedure of identification for multiple speech streams, listeners need to divide their attention to monitor multiple conversations, and then selectively attend to one speech stream while inhibiting the competing information.15 ) the age deficits in cognitive processing also contribute to speech - understanding difficulties in a multitalker conversation, possibly influenced by declined attentional capacity and inefficient allocation of attentional resources. as described above, all the age - related vascular, metabolic, or other systemic factors can cause detrimental changes to peripheral, central - auditory, or cognitive processes, yet various acoustic external factors such as fundamental frequency differences between competing voices can also impact the auditory segregation of collocated competing signals. the fundamental frequency (f0) is critical to speech perception especially in noise since f0 provides an important and robust cue to segregate multiple speech streams. the fundamental frequency means the frequency at which the vocal folds vibrate for making voiced speech sounds, and the vibration generates a periodic fluctuation of air pressure. f0 is calculated based on the number of vibrations per second and this is generally expressed in units of hertz (hz). f0 is inversely proportional to the vibrating mass and directly proportional to the tension (stiffness) of the vocal folds. depending on the features of mass or tension, a slow or fast vibration of vocal folds yields a low or high f0 value, consequently eliciting a low- or high - pitched sound. for example, the f0 value of men is typically an octave lower than that of women due to the longer and heavier vocal folds, eliciting a low - pitch sensation (e.g., f0 of 132 hz for men and 224 hz for women).17 ) f0 can be raised when a speaker increases tension to the vocal folds. the f0 value conveys not only the acoustic cues to vowel identity, intonation, and talker's gender, but also the cues on the speakers' age and even emotional state.16 ) when listeners simultaneously hear two speech signals with different f0s, it is easier to separate one from the other sound source. there is ample evidence that f0 separation remarkably improves performance for concurrent speech sounds overlapping in frequency and time, when the test materials were competing vowels,18 - 22 ) competing non - sense syllables,23 ) and competing sentences.16,24 - 26 ) a doubling of sound frequency is known as an octave. the octave interval is divided into twelve semitones (sts), which in one st corresponding to the 12th root of 2 (i.e.,1 st=1.0595). when concurrent vowels have been studied, often using a method referred to as the " double - vowel paradigm ", a small difference in f0 of less than 1 st dramatically enhanced identification accuracy or segregation perception. with f0 greater than 2 st, performance often reached an asymptote for normal - hearing (nh) listeners27 - 29 ) or both nh and hearing - impaired (hi) listeners.30 ) in contrast to the asymptote in the double - vowel paradigm, the young adults' identification of competing sentences received a gradual and progressive benefit from f0 shifts beyond 2 st, regardless of whether the natural variation of f0 over time was preserved or artificially removed.24,25,31,32 ) besides the robust benefit from f0 separation in young normal hearers, f0 benefit has also been found in hi listeners with cochlear pathology and in older adults.18,19,22,30,33 - 35 ) for example, the ability of 8 nh (age range : 21 - 30 yrs) and 9 hi (age range : 36 - 70 yrs) listeners to utilize f0s was compared in a monotonic double - vowel paradigm.18 ) in this experimental design, each vowel was made sufficiently audible to the hi group by a presentation level of at least 90 db spl. overall, nh participants outperformed the hi group in vowel identification, yet the amount of benefit from a f0 of 2 st was similar to each group (about 17 - 18 percentage points of improvement). the f0s greater than 2 st did not progressively enhance the vowel identification of either group. of masked vowel thresholds showed that both nh and hi groups could identify the weaker - intensity target vowel against the more intense masker vowel, regardless of f0 separation between vowel pairs. taken together, the poorer identification and greater masking in the hi group (compared to the nh group) led the authors to speculate that both would lead to less efficient use of f0 cues by the hi. as a series of studies,33 ) the double vowels were spectrally adjusted by introducing 25 db of gain at frequencies above 1000 hz and additionally tested six hi listeners (1 hi : 38 yrs, 5 hi : 53 - 69 yrs) who participated in the earlier study.18 ) despite high - frequency amplification ensuring the audibility of frequencies corresponding to the second and higher formants of vowels, the authors failed to observe a significant improvement in the overall identification performance of the hi group compared to that achieved without amplification. this suggests that the suprathreshold deficits in spectro - temporal processing, rather than speech audibility alone, contribute to the double - vowel performance of hi listeners. a follow - up study19 ) measured the ability to detect the presence of two different vowels and identify target words in young nh listeners and hi individuals of various ages (age range : 19 - 76 yrs). in this double - vowel paradigm, two experiments were conducted where f0, presentation mode, and target - to - masker ratio (tmr) at which listeners could just identify target vowels in the presence of a masking vowel were varied. of the first experiment showed that the hi listeners were less accurate at identifying the target vowels than the nh group across five tmrs, although a strong benefit from f0 of 2 st was observed in both groups. in the second experiment, both nh and hi groups were superior at using f0 cues in the dichotic condition compared to the monaural condition, with a greater dichotic benefit in the hi group than in the nh group. given the reduced overall ability of hi group in vowel identification of two experiments, the authors conjectured that the increased susceptibility to peripheral masking from broadened auditory filters would degrade the internal representations of competing vowels and consequently yield poorer double - vowel perception for the hi group. in another study,30 ) competing vowels were separated by f0 values from 0 to 9 st and also the vowels' harmonic structures were manipulated in order to explore whether the use of f0 depends on the disruption of harmonic structures in the low- or high - frequency regions. when the harmonic structures of five synthesized vowels were not disrupted, a f0 of 0.5 st improved the vowel - identification performance of the nh group by 33 percentage points and the same f0 separation benefited the hi group by 29 percentage points. neither group received additional benefit from f0 values beyond 1 st, which was consistent with the asymptote in other double - vowel studies. when the harmonic structures of double vowels were manipulated, the nh and hi groups were both effective at utilizing f0 cues in the lower - formant (f1) frequency region. however, the hi listeners inefficiently derived f0 information from higher - order harmonics (f2-f5). their findings supported a lack of negative effects of hearing loss when making use of the f0 cues, at least, in the low - frequency region, and thus suggested an association between the degraded use of f0 in the hi listeners and their reduced frequency selectivity at high frequencies. the previous studies described above18,19,30,33 ) only focused on the use of f0 in a double - vowel paradigm. very few studies measured identification of both concurrent vowels and sentences. for example, the performance of middle - aged and older listeners with nh (age range : 49 - 74 yrs) or with hearing impairment (age range : 59 - 77 yrs) was measured on three tasks.35 ) the three tasks tested were f0 discrimination of a single vowel and the identification of competing vowels and sentences. the for the difference limen of f0 (f0 dl) across five steady - state synthetic vowels revealed a greater sensitivity of nh listeners to f0 difference (0.5 - 2 hz for f0 dl) compared to the hi listeners (1 - 4 hz for f0 dl). when the subjects identified concurrent vowels, the f0 shift from 0 to 4 st provided the nh group with a perceptual benefit of 20 percentage points while the same f0 increased the mean identification accuracy of the hi group by only 8 percentage points. the authors reported not only the slightly less f0 benefit in the hi group compared to the nh group on the double vowels and double sentences, but also larger individual differences in performance of hi listeners. interestingly, a different factors appeared to be related to the large intersubject variability of hi listeners for three - task performances.35 ) hearing sensitivity of the hi individuals at 2000 hz was predictive of their sensitivity to the f0 differences on the discrimination task. in contrast, chronological age best accounted for the amount of f0 benefit in the competing vowel or sentence paradigms, indicating an association between age - related internal changes among the middle - aged and elderly participants and the higher - level processing required for the identification of competing speech signals. a weak link between double - vowel and double - sentence identification was also found, and this emphasized a possibility of overgeneralization of from competing - vowel studies to competing - sentence performance, at least for the middle - aged and older listeners. as noted, several previous studies18,19,30 ) agreed on the deleterious effect of cochlear hearing loss on performance in a double - vowel paradigm. in addition, there were often substantial age differences between the nh and hi groups. for example, the abilities between younger adults with normal hearing (ynh) and eldely listeners with normal hearing (enh) compared to demonstrate the age - related deficits in the use of f0 separation in a double - vowel paradigm.22 ) in the double - vowel paradigm, f0 dl measures using a synthesized vowel and vowel - identification performance using f0-separated double vowels were examined. when the f0 of the vowel /a/ was increased in steps of 0.1 hz under monaural presentation, the enh listeners were three times less sensitive to f0 compared to the ynh. here , individual differences in f0 dl were significantly correlated with age among the older adults. when the concurrent vowels were identified, mean identification accuracy of both listener groups improved markedly as f0 increased from 0 to 0.5 st in f0, yet not beyond a 0.5 st shift. moreover, the enh group was, on average, less accurate than the ynh group in identifying both vowels, yet the vowel identification of three enh individuals was comparable to the mean identification accuracy of the ynh group. analyses of error responses revealed that incorrect responses of both the ynh and the enh groups occurred more from the misidentification of one vowel than from both vowels, suggesting a presence of vowel dominance. based on the and the association between synchrony coding and benefit from f0 argued by de cheveign,36 ) the authors concluded that an age - related loss of neural synchrony increasing temporal jitter would evoke a declined periodicity coding and consequently a degraded benefit from f0 for concurrent speech signals. a recent study16 ) examined the negative effects of both hearing loss and age - related declines on the use of f0 by comparing four listener groups differing in hearing status and age. showed that f0 cues were beneficial to both cue - word detection and color - number identification performance. elderly adults with impaired hearing had the greatest difficulty with the identification task despite the application of spectral shaping to restore the audibility of the speech stimuli. also the authors concluded that the reduced audibility or aging alone is not the cause for the poor performance of the elderly hi listeners, indicating a combined contribution of hearing loss and aging on the reduced f0 benefit. as discussed above, age - related declines in the use of f0 appeared to be clear in numerous studies. can any hearing assistive devices help old people better access to f0 cues? recently, in order to address whether electro - acoustic and cochlear - implant hearing can enhance processing of f0, both young and older listeners' use of f0 cues was compared in simulations of electro - acoustic and cochlear - implant hearing.37 ) although the electric and acoustic stimulation (eas) is known to allow the listeners to combine residual low - frequency hearing with electrical hearing, expecting a greater benefit from f0s, some studies reported that old listeners may not benefit from eas devices as much as young listeners. thus, researchers37 ) focused on the ability of aged listeners in the processing of f0 cues using eas and cochlear - implant hearing simulations. the age - related deficits in the perception of vowel multiplicity were found in terms of amount of f0 benefit as well as overall identification. however, eas simulation provided a large inter - subject variability in f0 benefit of older individuals, consequently requiring more research on the effectiveness of eas stimulus to the old individuals in using f0 cues and other various acoustic cues. to understand the mechanism underlying the age - related degradation in the use of f0 or periodicity coding, it is critical to review the different aspects of three models, spectral, spectro - temporal, and temporal models, that can account for the f0 benefit observed for concurrent speech signals. in the spectral model, based on a place theory, auditory peripheral filtering is used to allocate frequency components corresponding to each competing vowel. according to the place theory of pitch, different pure tones produce maximal activity at different places with maximal activity near the base of the cochlea for high frequencies and near the apical part in the cochlea for low frequencies. these different spatial patterns of mechanical activity along the basilar membrane lead to activity in different auditory nerve fibers distributed along the length of the cochlea, producing the tonotopic responses of the auditory nerve. fig. 1 displays schematic magnitude spectra of mixtures of two different vowels, /i/, /a/, and mixed vowel of /i/ and /a/. as reported,38 ) the spectral envelope of the mixed vowels with different f0s shows peaks of each harmonic series at different frequencies corresponding to the first - formant (f1) frequency of each vowel, allowing a better resolution of individual harmonic structures. in contrast, the peaks in the spectral envelope of the mixed vowels with the same f0 are not separable as depicted by filled circles in fig. 1, suggesting that information on the individual harmonic series of each vowel would be poorly resolved for the competing vowels with the same f0. that is, in the spectral model based on the peripheral filtering, the degree of spectral resolution of two competing vowels was assumed to directly affect the identification accuracy of competing vowels, or the perceptual benefit of f0 in a double - vowel paradigm. in the spectro - temporal model, the information of each vowel is analyzed in a series of auditory peripheral channels as in the spectral model. yet, a subset of auditory channels responds to the period of one vowel and to the period of the other vowel. in this way, the periodicity corresponding to each vowel can be determined based on the spectro - temporal analysis over time even when the double vowels share the same auditory channel. when waveform interaction or beating is the basis of f0 benefit in the spectro - temporal model,21,28,39 ) a remarkable f0 benefit is possible even at small f0 separations between double vowels as long as the periodicity of the waveforms or the harmonicity of the speech signals is decoded.40 ) when there is a small f0 between two vowels, the f0 or corresponding harmonics of each vowel can be closely located in terms of frequency and possibly excite the same region of the basilar membrane. those two closely - spaced spectral or harmonic components might generate beating as a product of waveform interactions. 2a shows that a mixture of 125-hz and 128-hz pure tones produces rising and falling amplitude patterns or spectral envelopes with a repetition period of 3 hz, which is a relatively slow modulation. whereas, a mixture of 125-hz and 200-hz pure - tone (fig . when the temporal - envelope fluctuation of 3 hz is added to the dynamic fluctuation of two competing sounds, this might allow the listeners to glimpse the target signal against the masker one . better identification of longer - duration vowel pairs relative to shorter vowel pairs20,41,42) may be evidence of the spectro - temporal analyses since a longer duration might allow better encoding of a full modulation cycle in beating. as spectro - temporal model, an array of autocorrelation functions or autocorrelated temporal patterns between signals was also used to explain f0 benefit between concurrent vowels.43 ) in this spectro - temporal model based on autocorrelation segregation, autocorrelation functions of individual peripheral channel outputs are summed across channels. in a double - vowel paradigm, the largest peak within a summary of distributed autocorrelation function is considered a dominant periodicity, and all the channels with the dominant periodicity are selected to segregate a target vowel from a competing vowel. in other words, the auditory channels responding to the period of the dominant vowel are selected and partitioned from other channels, producing a strong f0 benefit in vowel identification. since the autocorrelation function determines the dominant periodicity or temporal pattern over the others, the requirement in this model is that each of the individual peripheral channels should be active for the different periodicities of each competing vowel, and the masker vowel should not dominate all the auditory channels. overall, the spectro - temporal models based on time - domain periodicity have been better than the spectral model in explaining the significant role of a very small f0 shift in the double - vowel paradigm, especially when the level of the target vowel is at or above that of the masker vowel. however, both the spectral model and the spectro - temporal model fail to explain how the f0 separation becomes beneficial to vowel identification even when the level of the target vowel is weaker, by 10 or 20 db, than that of the interfering vowel,19,36,44 ) presumably making all the auditory channels dominated by the more intense vowel and the individual harmonics of the target vowel unresolved. a temporal model based on a neural cancellation mechanism was proposed to explain the salient f0 benefit at negative tmrs more effectively.36 ) the temporal model based on the neural cancellation filter employs the temporal discharge pattern of auditory nerve fibers occurring within a channel, rather than requiring harmonic resolution across channels. fig. 3 demonstrates how the neural cancellation filter works by displaying a discharge distribution of an auditory never fiber in response to a 100-hz pure - tone pulse.36 ) the auditory - nerve fiber response to a single vowel with f0 of 100 hz is represented as a simple half - wave rectified sine wave of 100 hz in fig. 3a, and this serves as the input to the neural cancellation filter to the vowel. the neural cancellation filter calculates a delay that corresponds to the periodicity within input spikes and cancels the input spikes corresponding to the delay pattern, seen as the output of the cancellation filter in fig. 3c is another input consisting of a half - wave rectified sum of two sine waves, 100 hz (target) and 80 hz (competing), corresponding to a mixture of 100-hz target vowel and 80-hz masker vowel with a 10-db greater intensity. with competing vowels with different f0 values, the neural cancellation filter calculates a delay according to different periodicity of each vowel and is tuned to cancel the period of the masker vowel, discarding information about the periodicity of the masker vowel, as displayed in fig. the temporal model is based on the periodicity calculation, and this thus supposes that auditory system can derive pitch information from the period of discharges of auditory nerve fibers that do not resolve individual harmonics of the speech signal, even though the target vowel is presented with the more intense masker vowel. in other words, the temporal model selects the auditory channels based on the neural cancellation filters, not based on the outputs of auditory peripheral channels. this also appears consistent with some behavioral findings of a strong f0 benefit at unfavorable tmrs.19,44 ) using the guinea pig, the distribution of neural synchrony was examined across the population of auditory nerve fibers.45,46 ) by counting the spikes in bins synchronized with the double vowels, a histogram was constructed determining interspike interval distributions of the auditory nerve to the mixed vowel. the data were well fit via temporal analysis, compared to fits by spectral or spectro - temporal analyses. other animal studies, based on a wide variety of measurements, have observed an age - related reduction in the synchrony of auditory nerve fiber responses.47 - 51 ) as noted above, among the three types of model that have been developed, the temporal model provides the most favorable explanation for f0 benefit at low tmr values and for the age - related reduction in the f0 benefit for the vowel identification. if the older listeners have reduced neural activity or a loss of neural synchrony, this would in a decline in periodicity coding and an inefficient neural cancellation of competing speech signals. however, this does not necessarily mean that the deleterious effect of cochlear hearing loss would be unrelated to the outputs of the neural cancellation operation. although a broadening of auditory filters due to cochlear pathology would not directly damage the process of neural cancellation operations per se, the reduced input information due to the reduced audibility would, at least partially, degrade the input into the neural cancellation filter, possibly limiting the effectiveness of the neural cancellation filter. the primary objective of this study was to provide information on the age - related declines in the use of f0s between competing voices and the controversial theoretical explanations on this issue. as reviewed, aging adversely affected the identification of the target speech, either vowel or sentence, against interfering competing speech. as a possible explanation, aging can negatively influence neural encoding of the temporal properties in competing sounds, consequently ing in a declined periodicity coding and an inefficient neural cancellation of competing speech signals. this was better supported by temporal model based on the periodicity calculation compared to other theoretical models. in order to improve the ability of older adults to utilize f0s between competing voices, future studies are needed to investigate innovative technical development of the hearing assistive devices focusing on better access to the f0 segregation cues and also deriving greater f0 benefits to the older listeners.

a common complaint of older listeners is that they can hear speech, yet can not understand it, especially when listening to speech in a noise. when target and competing speech signals are concurrently presented, a difference in the fundamental frequency (f0) between competing speech signals, which determines the pitch of voice, can be an important and commonly occurring cue to facilitate the separation of the target message from the interfering message, consequently improving intelligibility of the target message. to address the question of whether the older listeners have reduced ability to use f0 and how the age - related deficits in the processing of f0 are theoretically explained, this paper is divided into three parts. the first part of this article summarizes how the speech - communication difficulties that older listeners have are theoretically explained. in the second part , literatures on the perceptual benefits from f0 and the age - related deficits on the use of f0 are reviewed. as a final part, three theoretical models explaining the general processing of f0 are compared to discuss which better explains the age - related deficits in the processing of f0.

carotid artery stenting (cas) is the standard modality for patients with severe carotid artery stenosis at high risk for carotid endarterectomy. however, a major drawback of cas is the potential risk of embolic stroke caused by the dislodgement of atheromatous plaque during the procedure. the traditional cas procedure involves initial vascular balloon dilatation (pre - dilatation) to achieve access for other devices, followed by optional placement of an embolic protection device (epd), positioning of a stent across the stenosis and, finally, post - stenting balloon dilatation (post - dilatation) to achieve maximum vessel expansion. emboli can arise in each procedural step, but most emboli are produced during the post - dilatation stage, which might occur if the balloon pushes the stent struts against the atheromatous plaque. post - dilatation is therefore considered to be the riskiest part of the procedure.1 2 to minimize procedural thromboembolic complications, we sometimes perform cas without post - dilatation; the aim of this study was to evaluate the efficacy and safety of our technical strategy for cas. a retrospective registry between may 2005 and april 2012 was accessed to recover 171 consecutive patients with 178 carotid artery stenoses who underwent cas with an epd at either the national hospital organization mito medical center (ibaraki, japan) or an affiliated hospital. cas was indicated in symptomatic or asymptomatic patients by a carotid artery stenosis of > 50% or > 80%, respectively, according to north american symptomatic carotid endarterectomy trial (nascet) criteria.3 our study included 169 patients (148 men, 21 women) with 176 carotid artery stenoses who underwent cas without post - dilatation. a total of 108 lesions (61.4%) were symptomatic and 68 (38.6%) were asymptomatic. patients received two of the three following antiplatelet agents: aspirin (100 mg), clopidogrel (75 mg) and cilostazol (200 mg) for at least 1 week before the procedure. systemic heparinization was administered following insertion of a sheath, and an activated clotting time of 300 s was maintained during the procedure. a guiding catheter or long sheath was advanced into the common carotid artery. under roadmap guidance, the stenotic lesion was crossed with an epd which was navigated distal to the stenotic lesion. all 107 cas procedures through may 2010 were performed using the guardwire temporary occlusion and aspiration system (medtronic, santa rosa, california, usa). after june 2010 we primarily performed cas using the filterwire ez embolic protection system (boston scientific, natick, massachusetts, usa). cas was performed using the guardwire system for patients with soft plaque deposits, as identified by a black blood mri indicating high - intensity and long lesions (> 25 mm ; figure 1). after june 2010 we performed cas using the filterwire ez system for 60 lesions and the guardwire system for nine lesions. pre - stenting balloon dilatation was subsequently applied to the stenotic lesions after epd placement and was usually performed using a 4.05.5 mm30 mm angioplasty balloon. the most commonly used stents were wallstents (boston scientific, natick, massachusetts, usa), which were applied in 174 lesions (98.9%), and precise stents (cordis, johnson & johnson, miami, florida, usa), which were applied in two lesions. angiographic views of the head and neck were obtained at the onset of the procedure and after successful stent placement. moreover, 16 patients with preoperative hemodynamic compromise or postoperative angiographic hyperemia of the lenticulostriate arteries underwent intravenous deep sedation for 24 h following the procedure. all the patients underwent a clinical examination (including a neurological examination) and mri including dwi both before and 24 h after the procedure. two antiplatelet agents were administered for a minimum of 3 months after surgery, after which one of the agents was discontinued. following discharge, the patients were examined and ultrasound follow - up studies were performed at 30 days, 3 months and then once every 3 months indefinitely. an octogenarian with symptomatic carotid artery stenosis who underwent carotid artery stenting using the guardwire system. (a) black blood t1-weighted mri showing a high - intensity plaque lesion in the right internal carotid artery (white arrow). (b) angiogram of the right common carotid artery showing severe stenosis of the internal carotid artery with a long lesion. (c) post - procedural angiogram of the right common carotid artery (pre - dilatation with 430 mm balloon and stent deployment with 1024 mm carotid wallstent) showing improvement of the internal carotid artery stenosis. a major adverse event was defined as any incidence of stroke, myocardial infarction or death within 30 days after cas. stroke was defined as a neurological deficit that persisted for > 24 h, whereas a minor stroke was defined as a new neurological deficit that resolved completely or returned to baseline within 30 days. a transient ischemic attack (tia) was defined as a new neurological deficit that persisted for < 24 h and completely resolved or returned to baseline. statistical analysis was performed to evaluate the efficacy of our cas procedure according to epd as well as the proportion of residual stenosis immediately after the procedure. nominal data were analyzed using the fisher exact test and numerical data were analyzed using the t test for parametric values or the mann a retrospective registry between may 2005 and april 2012 was accessed to recover 171 consecutive patients with 178 carotid artery stenoses who underwent cas with an epd at either the national hospital organization mito medical center (ibaraki, japan) or an affiliated hospital. cas was indicated in symptomatic or asymptomatic patients by a carotid artery stenosis of > 50% or > 80%, respectively, according to north american symptomatic carotid endarterectomy trial (nascet) criteria.3 our study included 169 patients (148 men, 21 women) with 176 carotid artery stenoses who underwent cas without post - dilatation. a total of 108 lesions (61.4%) were symptomatic and 68 (38.6%) were asymptomatic. patients received two of the three following antiplatelet agents: aspirin (100 mg), clopidogrel (75 mg) and cilostazol (200 mg) for at least 1 week before the procedure. systemic heparinization was administered following insertion of a sheath, and an activated clotting time of 300 s was maintained during the procedure. a guiding catheter or long sheath was advanced into the common carotid artery. under roadmap guidance, the stenotic lesion was crossed with an epd which was navigated distal to the stenotic lesion. all 107 cas procedures through may 2010 were performed using the guardwire temporary occlusion and aspiration system (medtronic, santa rosa, california, usa). after june 2010 we primarily performed cas using the filterwire ez embolic protection system (boston scientific, natick, massachusetts, usa). cas was performed using the guardwire system for patients with soft plaque deposits, as identified by a black blood mri indicating high - intensity and long lesions (> 25 mm ; figure 1). after june 2010 we performed cas using the filterwire ez system for 60 lesions and the guardwire system for nine lesions. pre - stenting balloon dilatation was subsequently applied to the stenotic lesions after epd placement and was usually performed using a 4.05.5 mm30 mm angioplasty balloon. the most commonly used stents were wallstents (boston scientific, natick, massachusetts, usa), which were applied in 174 lesions (98.9%), and precise stents (cordis, johnson & johnson, miami, florida, usa), which were applied in two lesions. angiographic views of the head and neck were obtained at the onset of the procedure and after successful stent placement. moreover, 16 patients with preoperative hemodynamic compromise or postoperative angiographic hyperemia of the lenticulostriate arteries underwent intravenous deep sedation for 24 h following the procedure. all the patients underwent a clinical examination (including a neurological examination) and mri including dwi both before and 24 h after the procedure. two antiplatelet agents were administered for a minimum of 3 months after surgery, after which one of the agents was discontinued. following discharge, the patients were examined and ultrasound follow - up studies were performed at 30 days, 3 months and then once every 3 months indefinitely. an octogenarian with symptomatic carotid artery stenosis who underwent carotid artery stenting using the guardwire system. (a) black blood t1-weighted mri showing a high - intensity plaque lesion in the right internal carotid artery (white arrow). (b) angiogram of the right common carotid artery showing severe stenosis of the internal carotid artery with a long lesion. (c) post - procedural angiogram of the right common carotid artery (pre - dilatation with 430 mm balloon and stent deployment with 1024 mm carotid wallstent) showing improvement of the internal carotid artery stenosis. a major adverse event was defined as any incidence of stroke, myocardial infarction or death within 30 days after cas. stroke was defined as a neurological deficit that persisted for > 24 h, whereas a minor stroke was defined as a new neurological deficit that resolved completely or returned to baseline within 30 days. a transient ischemic attack (tia) was defined as a new neurological deficit that persisted for < 24 h and completely resolved or returned to baseline. statistical analysis was performed to evaluate the efficacy of our cas procedure according to epd as well as the proportion of residual stenosis immediately after the procedure. nominal data were analyzed using the fisher exact test and numerical data were analyzed using the t test for parametric values or the mann cas was performed successfully for all lesions, although major adverse events occurred in four patients (2.3%). the overall stroke rate was 2.3% within 30 days after cas; none of the patients experienced myocardial infarction and there were no deaths. cerebral infarction and intracranial hemorrhage occurred in two patients each (1.1%) and tia occurred in three patients (1.7%). dwi obtained after cas revealed a high - intensity area in 26 of 176 procedures (14.8%). in 68 asymptomatic patients, one minor stroke (1.5%) occurred because of subacute instent thrombosis, one tia (1.5%) occurred within 30 days after cas and dwi obtained 24 h after cas revealed a high - intensity area in seven procedures (10.3%). in 108 symptomatic patients, one minor stroke (0.9%) occurred because of thromboembolic complications. two intracranial hemorrhages and two tias (1.9%) occurred within 30 days after cas, and dwi obtained 24 h after cas revealed a high - intensity area in 19 (17.6%) procedures (table 1). subacute in - stent thrombosis complicated with tia developed in one of the symptomatic patients. follow - up data beyond 1 year for all 176 lesions included two cases of symptomatic restenosis. the ipsilateral stroke rate was 1.1% (symptomatic 1.9% ; asymptomatic 0%) after 31 days. four asymptomatic restenosis cases were diagnosed during follow - up ultrasound examinations; subsequently, three of these patients underwent angioplasty and the overall restenosis rate was 3.4%. the mean stenosis diameter according to the nascet criteria3 was reduced from 75.011.8% to 17.311.2%. the immediate angiographic outcomes after cas were as follows: residual stenosis 10% (n=54, 30.7%); residual stenosis > 10% and 20% (n=52, 29.5%); residual stenosis > 20% and 30% (30% and 40% ( n=22, 12.5%); and residual stenosis > 40% (n=5, 2.8%). restenosis or subacute in - stent thrombosis after cas was observed in eight patients. the mean stenosis rate immediately after cas was 32.1% in this group, compared with 16.9% in the group without restenosis or subacute in - stent thrombosis. thus, the mean stenosis rate immediately after cas was significantly higher in the group with restenosis or subacute in - stent thrombosis (p=0.0004). five of 27 patients (18.5%) with residual stenosis immediately after cas > 30% suffered restenosis or in - stent thrombosis and three of 149 patients (2.0%) without residual stenosis immediately after cas > 30% suffered restenosis or in - stent thrombosis. the rate of restenosis or in - stent thrombosis was significantly higher in the group with residual stenosis immediately after cas > 30% (table 2). comparison of the procedural outcomes according to the residual stenosis immediately after carotid artery stenting (cas) a comparison of epd (table 3) revealed no difference in the incidence of stroke within 30 days after cas and high intensity areas on dwi obtained 24 h after cas. comparison of the procedural outcomes between patients treated with the guardwire (gw) and filterwire ez (fw) systems dwi, diffusion - weighted imaging. strokes arising from carotid stenosis are most often due to atheroembolisms.46 during carotid endarterectomy the plaque is completely removed; however, with carotid stenting, the plaque remains contained between the stent and the vessel wall. stroke occurring after cas is probably caused by the release of fractured plaque deposits through the struts of the stent. in the carotid revascularization endarterectomy versus stenting trial,7 the periprocedural stroke rate was significantly higher in the stenting group than in the endarterectomy group (4.1% vs 2.3%, p=0.01). transcranial doppler studies have demonstrated the generation of emboli with each passage across a stenosis with a guidewire, epd, balloon or stent,8 9 with the highest potential for embolization occurring during post - dilatation when the balloon crushes friable plaque against the metal stent struts.8 9 although the clinical significance of microemboli is unclear, ackerstaff et al10 demonstrated in a study of 550 patients that multiple microemboli (> 5 showers) at post - dilatation were independently associated with cerebral deficits. the traditional cas technique includes initial balloon pre - dilatation during which the epd or stent is accessible distal to the stenosis, subsequent optional placement of the epd followed by deployment of a stent covering the stenosis, and finally post - dilatation to achieve maximum vessel expansion after stent deployment. we performed cas without post - dilatation to minimize plaque disruption and the incidence of periprocedural stroke. the of this study revealed a relatively low incidence of complications within 30 days after the procedure (2.3% overall stroke, death or myocardial infarction). symptomatic patients are known to have emboligenic plaque.11 in the carotid revascularization endarterectomy versus stenting trial, the rate of any post - procedural stroke within 30 days was 5.5% in symptomatic patients treated with cas and 3.2% in symptomatic patients treated with carotid endarterectomy.7 in this study, the rate of any post - procedural stroke within 30 days was 1.5% in asymptomatic patients and 2.8% in symptomatic patients. in symptomatic patients, the incidence of periprocedural stroke was relatively low in this study because the omission of post - dilatation might have minimized plaque disruption and reduced thromboembolic complications. most embolic complications occur after the procedure and are probably caused by late emboli through the struts of the stent, as described above.11 a closed cell stent has smaller struts than an open cell stent.12 bosiers et al11 reported that the rate of post - procedural events was higher when using the open cell stent than when using the closed cell stent. these differences were highly pronounced among symptomatic patients.11 wallstents, the most commonly used stents in this study, were applied in 174 lesions (98.9%). in addition to the omission of post - dilatation, the use of a wallstent might also reduce thromboembolic complications. the closed cell stent has a lower radial force than an open cell stent,12 and poor stent expansion immediately after the procedure was associated with a higher risk of restenosis.13 furthermore, omission of post - dilatation and the use of a closed cell stent might cause restenosis. a systematic review by grschel et al14 reported a restenosis rate with conventional cas of 46% at 1 year; the rate of restenosis with our cas protocol (3.4%) was therefore lower than that previously reported. in our procedure, the goal of pre - dilatation was to enlarge the stenotic lumen suboptimally with only the balloon. therefore, pre - dilatation alone without post - dilatation can achieve a sufficient luminal opening to prevent restenosis. on the other hand, arterial injury with both balloons and stents can initiate an inflammatory response and activate a proliferative repair process, the end of which can lead to luminal narrowing and in - stent restenosis.1519 in fact, omission of post - dilatation and the use of a closed cell stent might be beneficial to minimize such restenosis incidents. on the other hand, the rate of restenosis or in - stent thrombosis was significantly higher in the patient group with > 30% residual stenosis immediately after cas in the present study. post - dilatation might therefore be added to avoid restenosis and in - stent thrombosis in patients with > 30% residual stenosis. nevertheless, additional post - dilatation might induce thromboembolic complications, particularly in symptomatic patients with unstable risky plaque deposits. in cases with > 30% residual stenosis immediately after cas , we should consider whether or not to add post - dilatation to the procedure for symptomatic patients and decide how much post - dilatation to add. in a previous study to evaluate microembolic signals detected by transcranial doppler during cas, the amount of microembolic signals was significantly higher using the filterwire system than with the guardwire system.20 the distal occlusion device is thought to be more effective than the filter in reducing distal embolization.20 however, balloon occlusion (whether proximal or distal) is associated with cerebral intolerance in 510% of cases.21 22 on the other hand, the main advantage of filters is the maintenance of blood flow to the brain throughout the procedure. a comparison of epds revealed no difference in the incidence of stroke within 30 days after cas and the high - intensity area on dwi obtained 24 h after cas in the present study. cas without post - dilatation can take advantage of filter protection because of the reduction in distal embolisms. second, we could not observe any advantage of omission of post - dilatation using the open cell stent, although closed cell stents were used in 174 lesions (98.9%) while open cell stents were used in only two lesions. lastly, this analysis could be improved by a longer radiological and clinical follow - up period to further evaluate the clinical significance. carotid stenting without post - dilatation might achieve minimal plaque disruption and reduction of thromboembolic complications. our cas procedure is effective, particularly in symptomatic patients. although it might be possible to employ new types of stents and epds in the future, concepts to minimize plaque disruption are important because plaque deposits remain contained in the vessel wall in carotid stenting procedures

purposeto evaluate the clinical outcome and mri findings after carotid artery stenting (cas) without post-dilatation.methodsbetween may 2005 and april 2012, a total of 169 consecutive patients (61.4% symptomatic) underwent 176 cas procedures performed with an embolic protection device (guardwire, n=116 ; filterwire ez, n=60). all stents were deployed without post - dilatation. periprocedural complications and mid - term outcomes were analyzed.the stroke rate was 2.3% within 30 days post - cas (asymptomatic patients 1.5% ; symptomatic patients 2.8%). cerebral infarction occurred in one asymptomatic patient (1.5%) and one symptomatic patient (0.9%). intracranial hemorrhage occurred in two symptomatic patients (1.9%). post - cas diffusion - weighted imaging (dwi) revealed a high - intensity area in 26 of 176 procedures (14.8%). ipsilateral stroke after 31 days occurred in two patients (1.1%) and restenosis occurred in six (3.4%). a post - cas comparison of the embolic protection devices revealed no difference in stroke incidence within 30 days and in dwi high - intensity area.our cas procedure without post - dilatation is feasible, safe and associated with a low incidence of stroke and restenosis.

intracerebral hemorrhage (ich) is a common cerebrovascular disease with a high mortality and poor outcomes in clinical practice, and also the leading cause of death and disability in elderly patients.1 it was reported to account for about 10% of all strokes and 30% of all cerebrovascular diseases.2,3 the mortality rate could be up to 50%, and the mortality rate in one month could be up to 40%.3,4 moreover, there could be up to 40% of severely disabled survivors.3 normally, the edema and hematoma expansion are the two major factors contributing to worsened outcomes and secondary damage.5 many factors could lead to ich, but hypertension is the major cause of ich, and the hypertensive ich (hich) is a common neurological disease. to date, there are still no effective drug therapies for hich.6 moreover, hich is characterized by high incidence, high mortality and high morbidity, which seriously endangers the health of patients and causes substantial economic burden for families and society. therefore, it is urgently necessary to develop an effective therapy for this disease. during the treatment, the key point is to quickly clear the hematoma and reduce the intracranial pressure.7 along with minimally invasive technique, minimally invasive surgery (mis) for hich is gaining increasing attention. it could effectively decrease the mortality rate in the acute treatment of patients with hich.8 additionally, some previous studies reported that the mild hypothermia therapy (mht) could be useful for neuroprotection and be used to treat cerebral edema after acute brain injury.911 previous study reported that the addition of mht could significantly improve the efficacy of mis in treating hich.12 therefore, the aim of this study was to review the available published studies and conduct a meta - analysis to systematically assess whether the combined application of mis and local mht (lmht) (mis+lmht) could in a better efficacy than mis alone. the first step of this meta - analysis was to obtain the eligible clinical trials. we conducted electronic searches in the following databases: cochrane controlled trails register, pubmed, embase, web of science, chinese biomedical literature database on disc and chinese national knowledge infrastructure (dated up to september 2016). the search terms that we used were hypothermia , minimally invasive and hypertensive cerebral hemorrhage. the inclusion criteria included the following items: 1 ) randomized clinical trials comparing the mis+lmht with mis alone; 2 ) patients with hypertensive cerebral hemorrhage over 18 years of age; 3 ) the outcomes including at least one of these three indexes: response rate, mortality rate and neurologic function; and 4 ) patients could provide informed consent. the exclusion criteria included the following items: 1 ) no control group or not used mis as the control group and 2 ) case reports, reviews and duplicate studies. the neurologic function, barthel index and side effects were chosen as the secondary outcomes. the barthel index was used to assess the activities of daily living (adl) after treatment. because pneumonia might be the main side effect caused by hypothermia,13 we only analyzed the incidence of pneumonia to assess the acceptability of these treatment methods. in order to ensure the high accuracy of the extracted data, two authors (yu han and ke sheng) were arranged to independently screen the potential studies according to the aforementioned inclusion / exclusion criteria and extract the data. the extracted data included the demographic data of the patients, the parameters of the mis and lmht, the first author and outcomes (primary and secondary outcomes). good - faith efforts were made to obtain the data which were not available from the included studies. revman 5.1 software was used to conduct the meta - analysis. for discontinuous data, the summary odds ratio (or) was used as the effect size; for continuous data, the weighted mean difference (wmd) was used as the effect size. the chi - square test and i index were used to assess the heterogeneity.14 if the corresponding p - value was more than 0.10 and i was less than 50%, then the mantel haenszel fixed - effects model was used; otherwise, the random - effects model was used. the first step of this meta - analysis was to obtain the eligible clinical trials. we conducted electronic searches in the following databases: cochrane controlled trails register, pubmed, embase, web of science, chinese biomedical literature database on disc and chinese national knowledge infrastructure (dated up to september 2016). the search terms that we used were hypothermia , minimally invasive and hypertensive cerebral hemorrhage. the inclusion criteria included the following items: 1 ) randomized clinical trials comparing the mis+lmht with mis alone; 2 ) patients with hypertensive cerebral hemorrhage over 18 years of age; 3 ) the outcomes including at least one of these three indexes: response rate, mortality rate and neurologic function; and 4 ) patients could provide informed consent. the exclusion criteria included the following items: 1 ) no control group or not used mis as the control group and 2 ) case reports, reviews and duplicate studies. the response rate was defined according to the criteria of the included studies. the neurologic function, barthel index and side effects were chosen as the secondary outcomes. the barthel index was used to assess the activities of daily living (adl) after treatment. because pneumonia might be the main side effect caused by hypothermia,13 we only analyzed the incidence of pneumonia to assess the acceptability of these treatment methods. in order to ensure the high accuracy of the extracted data, two authors (yu han and ke sheng) were arranged to independently screen the potential studies according to the aforementioned inclusion / exclusion criteria and extract the data. the extracted data included the demographic data of the patients, the parameters of the mis and lmht, the first author and outcomes (primary and secondary outcomes). good - faith efforts were made to obtain the data which were not available from the included studies. discontinuous data, the summary odds ratio (or) was used as the effect size; for continuous data, the weighted mean difference (wmd) was used as the effect size. the chi - square test and i index were used to assess the heterogeneity.14 if the corresponding p - value was more than 0.10 and i was less than 50%, then the mantel haenszel fixed - effects model was used; otherwise, the random - effects model was used. based on the inclusion and exclusion criteria, 129 studies were excluded. the reasons for exclusion included the following: 1 ) no randomization, 2 ) no control group, 3 ) compared the mis+lmht with the conservative treatment, 4 ) no available data, 5 ) did not use mis and 6 ) being a retrospective study. finally, 28 clinical trials composed of 2,325 adult patients with hypertensive cerebral hemorrhage were included in this meta - analysis.1643 the matched demographic data were observed in the included studies (table 1). more than half of the included studies used cooling blanket as the device of lmht. overall, 916 of 1,156 (79.2%) patients receiving the mis+lmht and 673 of 1,129 (59.6%) patients receiving mis alone were classified as responders. the pooled or was 2.68 with 95% confidence interval (ci)=2.223.24, which indicated that the mis+lmht could yield a higher response rate than mis alone. overall, 79 of 883 (8.9%) patients receiving the mis+lmht and 162 of 868 (18.7%) patients receiving mis alone died after treatment. the pooled or was 0.43 with 95% ci=0.320.57, which indicated that the mis+lmht could yield a lower mortality rate than mis alone. the funnel plot showed that there was no publication bias (figure 2). among the included studies, 11 studies used css to assess the neurologic function of patients (figure 4). eight of 11 studies assessed the neurologic function one month after treatment; two studies conducted the assessment 21 days later and one study 14 days later. the pooled wmd was 5.83 with 95% ci=7.18 to 4.47, which indicated that the mis+lmht could be more effective than mis alone in improving the neurologic function of patients. seven studies assessed adl using barthel index one month after treatment (figure 5a). heterogeneity (i=67%, p=0.006) existed. ten studies assessed adl three months after treatment (figure 5b). heterogeneity (i=87%, p<0.00001) existed. these indicated that the mis+lmht could be more effective than mis alone in improving the adl of patients. overall, 17 of 77 patients receiving the mis+lmht and 16 of 76 patients receiving the mis alone experienced pneumonia. the pooled or was 1.05 with 95% ci=0.432.59, which indicated that the incidence of pneumonia was similar between these two treatment methods. based on the inclusion and exclusion criteria, 129 studies were excluded. the reasons for exclusion included the following: 1 ) no randomization, 2 ) no control group, 3 ) compared the mis+lmht with the conservative treatment, 4 ) no available data, 5 ) did not use mis and 6 ) being a retrospective study. finally, 28 clinical trials composed of 2,325 adult patients with hypertensive cerebral hemorrhage were included in this meta - analysis.1643 the matched demographic data were observed in the included studies (table 1). more than half of the included studies used cooling blanket as the device of lmht. overall, 916 of 1,156 (79.2%) patients receiving the mis+lmht and 673 of 1,129 (59.6%) patients receiving mis alone were classified as responders. the pooled or was 2.68 with 95% confidence interval (ci)=2.223.24, which indicated that the mis+lmht could yield a higher response rate than mis alone. overall, 79 of 883 (8.9%) patients receiving the mis+lmht and 162 of 868 (18.7%) patients receiving mis alone died after treatment. the pooled or was 0.43 with 95% ci=0.320.57, which indicated that the mis+lmht could yield a lower mortality rate than mis alone. among the included studies, 11 studies used css to assess the neurologic function of patients (figure 4). eight of 11 studies assessed the neurologic function one month after treatment; two studies conducted the assessment 21 days later and one study 14 days later. heterogeneity (i=91%, p<0.0001) existed. the pooled wmd was 5.83 with 95% ci=7.18 to 4.47, which indicated that the mis+lmht could be more effective than mis alone in improving the neurologic function of patients. seven studies assessed adl using barthel index one month after treatment (figure 5a). heterogeneity (i=67%, p=0.006) existed. ten studies assessed adl three months after treatment (figure 5b). heterogeneity (i=87%, p<0.00001) existed. these indicated that the mis+lmht could be more effective than mis alone in improving the adl of patients. overall, 17 of 77 patients receiving the mis+lmht and 16 of 76 patients receiving the mis alone experienced pneumonia. the pooled or was 1.05 with 95% ci=0.432.59, which indicated that the incidence of pneumonia was similar between these two treatment methods. we conducted this meta - analysis of 28 randomized clinical trials to compare the efficacy of mis+lmht with mis alone in the treatment of patients with hich. the showed that the mis+lmht could yield higher response rate (or=2.68) and lower mortality rate (or=0.43). moreover, this treatment modality could significantly improve the neurologic function and adl of patients. with respect to the analysis of side effects, only three studies reported the number of patients with pneumonia, which was insufficient to make a robust on the safety of the mis+lmht. however, these demonstrated that the lmht could be an effective augmentation strategy for mis in treating patients with hich. in this study, almost all relevant randomized clinical trials were included, but some trials might have been missed, partly because these were published in some journals that are not indexed by international databases. fortunately, it is likely that these trials are of low quality, and could not significantly affect the .44 additionally, there was one trial without the needed data,45 and despite our best efforts, we could not obtain them. however, this trial concluded that the mis+lmht could be good at protecting nerve cell and promoting neurofunctional rehabilitation. therefore, this trial would not affect our . under conditions of mild hypothermia, the oxygen consumption and metabolic rate in brain tissue are decreased, the production of free radicals is reduced and the synthesis of xanthine oxidase is slowed down and then the tissue damage could be alleviated. meanwhile, the abnormal sodium calcium exchange between cell membrane and sarcoplasmic reticulum is suppressed, which could alleviate the calcium overload.46 under normal conditions, vasoactive substances, such as endothelin and vascular vasopressin, are in dynamic equilibrium to maintain the systolic and diastolic function of blood vessels.47 the acute period of ich is usually accompanied by the disorder of vasoactive substances. previous studies reported that the mild hypothermia could reduce the level of endothelin and vascular vasopressin.40,48 previous studies on ischemic and hemorrhagic stroke subtypes showed that the mild hypothermia (body temperature reduced by 3c5c) was neuroprotective.49,50 but one point should be noticed in clinical practice: systemic mild hypothermia is difficult to perform because of its possible side effects.10,11 alternatively, local mild hypothermia could quickly obtain the target temperature and overcome the potential side effects.51 therefore, compared to systemic mild hypothermia, the local mild hypothermia could be more effective in treating patients with hich. first, all of the included studies were from the people s republic of china, which might limit the applicability of our findings.52 second, only three studies were used to analyze the side effects; hence, future studies are needed to further assess the safety of the mis+lmht. third, the target temperature and treatment time of lmht were not consistent, but there were also the general problems for meta - studies to solve.53,54 by pooling analysis of 28 randomized clinical trials, we found that the addition of lmht could significantly improve the efficacy of mis in the treatment of patients with hich. the or of response rate and mortality rate was 2.68 and 0.43, respectively, which was in favor of the mis+lmht. the clinical applicability of this modality showed greater promise and should be further explored and optimized.

previous studies reported that the mild hypothermia therapy (mht) could significantly improve the clinical outcomes for patients with hypertensive intracerebral hemorrhage (hich). therefore , this meta - analysis was conducted to systematically assess whether the addition of local mht (lmht) could significantly improve the efficacy of minimally invasive surgery (mis) in treating hich.methodsrandomized clinical trials on the combined application of mis and lmht (mis+lmht) vs mis alone for treating hich were searched up to september 2016 in databases. response rate and mortality rate were the primary outcomes, and the neurologic function and barthel index were the secondary outcomes. side effects were also analyzed.totally, 28 studies composed of 2,325 patients were included to compare the efficacy of mis+lmht to mis alone. the therapeutic effects of mis+lmht were significantly better than mis alone. the pooled odds ratio of response rate and mortality rate was 2.68 (95% confidence interval =2.223.24) and 0.43 (95% ci=0.320.57), respectively. in addition, the mis+lmht led to a significantly better improvement in the neurologic function and activities of daily living. the incidence of pneumonia was similar between the two treatment methods.these indicated that compared to mis alone, the mis+lmht could be more effective for the acute treatment of patients with hich. this treatment modality should be further explored and optimized.

a total of 13 women and 8 men participated in the first study (lipid study). briefly, the individuals were normal weight (bmi 23.7 0.8 kg / m), were aged 26.4 0.8 years, had a waist - to - hip ratio (whr) of 0.83 0.02, and had basal lipid parameters in the normal range (total cholesterol 4.53 0.17 mmol / l, hdl cholesterol 1.44 0.03 mol / l, ldl cholesterol 2.64 0.14 mmol / l, triacylglycerols 1.01 0.08 mmol / l, and ffas 0.68 0.12 none of the participants had taken any medication for at least 3 months before the study . all participants were initially screened for any systemic disease or biochemical evidence of impaired hepatic or renal function . subjects with a history of hypertension, type 2 diabetes, renal or liver disease, dyslipidemia, heart failure, or a family history of diabetes or any other endocrine disorder were excluded from this study . body weight of the participants was stable for at least 2 months before the study . informed written consent was obtained from each participant . for the second human trial ( ppar study) 7 male and 10 female volunteers with impaired glucose tolerance the individuals were overweight (bmi 32.8 2.2 kg / m), were aged 59.1 1.6 years, and had a whr of 0.88 0.03. all participants were initially screened for any systemic disease or biochemical evidence of impaired hepatic or renal function. patients with heart failure, impaired hepatic or renal function, anemia, disturbed coagulation, or any other endocrine disorder were excluded. informed written consent was obtained from all subjects after explanation of the nature, purpose, and potential risks of the studies. the patients were instructed not to modify their way of living (diet and exercise) during the treatment period. all study protocols were approved by the institutional review board of the charit medical school, campus benjamin franklin. hepg2 cells were grown in eagle's minimum essential medium with 10% fcs and 1% nea; 24 h before analysis, hepg2 cells were incubated with basal serum - free media. to analyze the effect of ffas, palmitic, oleic, or linoleic acid (sigma - aldrich , seelze, germany) or a mixture of these fatty acids (one - third of each) were dissolved in water with 125 mol / l naoh and 10 mol / l fatty acid free bovine serum albumin (bsa) (calbiochem, germany) in a concentration of 160 mol / l (ratio 16:1) and cells were incubated within that medium for 1, 2, 4, 8, and 24 h. time course and concentration - dependent effects of ffas on fgf-21 secretion and expression were analyzed. therefore, primary human myoblasts were prepared from human skeletal muscle of healthy individuals und cultured in skeletal muscle cell growth medium (promocell, heidelberg, germany) containing 10% fetal bovine serum and 1.5% glutamine and gentamicin. after the first passage, myoblasts were separated from fibroblasts by neural cell adhesion molecule labeled magnetic beads (miltenyi biotech, bergisch - gladbach, germany). to analyze the effect of lecithin and glycerol on fgf-21 secretion , hepg2 cells were incubated for 4 h using a concentration of 2.5 mg / ml lecithin (fluka ; sigma - aldrich) and 1 mmol / l glycerol (sigma - aldrich). small - interfering rna (sirna) experiments were performed to investigate whether those effects depend on ppar. therefore, sirna for ppar (nos . a total of 100 pmol / well of each sirna and electroporation with amaxa nucleofector ii ( lonza cologne, cologne, germany) was used for transfection. the fgf-21 levels were measured in the supernatant by radioimmunoassay (phoenix europe, karlsruhe, germany) using 125 i labeled fgf-21 as a tracer and normalized on total protein levels. protein quantification was performed using the roti - nanoquant (karl - roth, karlsruhe, germany). for rt - pcr 20 ng of total rna per well were used and fgf-21 rna was analyzed according to the manufacturer's the instructions of the iscript sybr green rt - pcr kit (biorad, hercules, ca). real - time quantitative pcr was performed using an abi prism 7300 real - time pcr system (applied biosystems, foster city, ca). primer sequences will be sent upon request. the ddct - method (change in dct) eligible participants were randomly assigned to receive either lipid / heparin infusion (lhi) or saline / heparin infusion (shi) in a cross - over design for the lipid study. the study was performed during the early follicular phase of two subsequent menstrual cycles (days 46). to avoid interactions between the study procedures, the study was performed at intervals of at least 2 days in men. following a 10-h overnight fast , a short polyethylene catheter was inserted into an antecubital vein for infusion of test substances at 0800 h. another catheter was placed into the contralateral forearm vein for blood sampling. a 0.9% saline infusion plus heparin (0.4 units kg min) or a lipid infusion (abbolipid 20% , abbott, wiesbaden, germany ; or lipovens 20% , fresenius kabi, bad homburg, germany) plus heparin (0.4 units kg min) was infused at a constant rate of 1.5 ml / min for 330 min. four hours after the start of the saline / heparin infusion a hyperinsulinemic - euglycemic clamp was started in six subjects. each subject was studied before and after an 8-week rosiglitazone treatment (2 8 mg / day ; glaxosmithkline, mnchen, germany) using an oral glucose tolerance test and a hyperinsulinemic - euglycemic clamp as described previously. in brief, 40 miu / m per min human insulin (actrapid ; novo nordisk, bagsvaard, denmark) and a variable infusion of 10% glucose (serag wiessner, naila, germany) was used, and capillary glucose concentration was monitored every 5 min and was maintained between 4.0 and 4.9 mmol / l via variation of the glucose infusion rate. insulin sensitivity was assessed as m value and was calculated by dividing the average glucose infusion rate (mg glucose / min) during the steady state of the clamp by the body weight. plasma fgf-21 levels were measured before and during lhi shi, and steady state at the end of the hyperinsulinemic - euglycemic clamp. metabolic parameters, including ffas, triacylglycerols, total cholesterol, ldl cholesterol, hdl cholesterol, glucose, and insulin were determined as described previously. serum fgf-21 levels were determined by radioimmunoassay (phoenix europe) using 125 i labeled fgf-21 as tracer. the linear range of the assay was 0.58.5 ng / ml, and the standard range was 0.23430 ng / ml. the interassay and intra - assay coefficients of variation were 5.0 and 14%, respectively. all statistical procedures were performed using spss version 15.0 (spss, chicago, il). profiles of ffas, insulin, glucose, triacylglycerols, and fgf-21 were compared by repeated - measures anova with treatment and time as within - subject factors and the huynh - feldt- procedure as correction factor, which corrects for a positive biased f test for within - subject factors. the area under the concentration time curve (auc) was calculated by using the trapezoidal integration. the change in fgf-21, ffas, glucose, triacylglycerols and insulin between baseline and after lhi and shi was calculated by subtracting the baseline value from the value 4 h after each infusion procedure. data were compared by paired student's t test for normally distributed data and wilcoxon test for skewed data. the correlations between the values were estimated by pearson's correlation test and adjusted for age, sex bmi, and change in insulin. the unpaired t test or mann - whitney test were considered to be significant if the two - sided was < 0.05. hepg2 cells were grown in eagle's minimum essential medium with 10% fcs and 1% nea; 24 h before analysis, hepg2 cells were incubated with basal serum - free media. to analyze the effect of ffas, palmitic, oleic, or linoleic acid (sigma - aldrich , seelze, germany) or a mixture of these fatty acids (one - third of each) were dissolved in water with 125 mol / l naoh and 10 mol / l fatty acid free bovine serum albumin (bsa) (calbiochem, germany) in a concentration of 160 mol / l (ratio 16:1) and cells were incubated within that medium for 1, 2, 4, 8, and 24 h. time course and concentration - dependent effects of ffas on fgf-21 secretion and expression were analyzed. therefore, primary human myoblasts were prepared from human skeletal muscle of healthy individuals und cultured in skeletal muscle cell growth medium (promocell, heidelberg, germany) containing 10% fetal bovine serum and 1.5% glutamine and gentamicin. after the first passage, myoblasts were separated from fibroblasts by neural cell adhesion molecule labeled magnetic beads (miltenyi biotech, bergisch - gladbach, germany). to analyze the effect of lecithin and glycerol on fgf-21 secretion , hepg2 cells were incubated for 4 h using a concentration of 2.5 mg / ml lecithin (fluka ; sigma - aldrich) and 1 mmol / l glycerol (sigma - aldrich). the fgf-21 levels were measured in the supernatant and normalized to total protein levels. small - interfering rna (sirna) experiments were performed to investigate whether those effects depend on ppar. therefore, sirna for ppar (nos . a total of 100 pmol / well of each sirna and electroporation with amaxa nucleofector ii ( lonza cologne, cologne, germany) was used for transfection. the fgf-21 levels were measured in the supernatant by radioimmunoassay (phoenix europe, karlsruhe, germany) using 125 i labeled fgf-21 as a tracer and normalized on total protein levels. protein quantification was performed using the roti - nanoquant (karl - roth, karlsruhe, germany). for rt - pcr 20 ng of total rna per well were used and fgf-21 rna was analyzed according to the manufacturer's the instructions of the iscript sybr green rt - pcr kit (biorad, hercules, ca). real - time quantitative pcr was performed using an abi prism 7300 real - time pcr system (applied biosystems, foster city, ca). the ddct - method (change in dct) was used for relative quantification. fold changes in expression eligible participants were randomly assigned to receive either lipid / heparin infusion (lhi) or saline / heparin infusion (shi) in a cross - over design for the lipid study. the study was performed during the early follicular phase of two subsequent menstrual cycles (days 46). to avoid interactions between the study procedures, the study was performed at intervals of at least 2 days in men. following a 10-h overnight fast, a short polyethylene catheter was inserted into an antecubital vein for infusion of test substances at 0800 h. another catheter was placed into the contralateral forearm vein for blood sampling. a 0.9% saline infusion plus heparin (0.4 units kg min) or a lipid infusion (abbolipid 20% , abbott, wiesbaden, germany ; or lipovens 20% , fresenius kabi, bad homburg, germany) plus heparin (0.4 units kg min) was infused at a constant rate of 1.5 ml / min for 330 min. four hours after the start of the saline / heparin infusion a hyperinsulinemic - euglycemic clamp was started in six subjects. each subject was studied before and after an 8-week rosiglitazone treatment (2 8 mg / day ; glaxosmithkline, mnchen, germany) using an oral glucose tolerance test and a hyperinsulinemic - euglycemic clamp as described previously. in brief, 40 miu / m per min human insulin (actrapid ; novo nordisk, bagsvaard, denmark) and a variable infusion of 10% glucose (serag wiessner, naila, germany) was used, and capillary glucose concentration was monitored every 5 min and was maintained between 4.0 and 4.9 insulin sensitivity was assessed as m value and was calculated by dividing the average glucose infusion rate (mg glucose / min) during the steady state of the clamp by the body weight. eligible participants were randomly assigned to receive either lipid / heparin infusion (lhi) or saline / heparin infusion (shi) in a cross - over design for the lipid study. the study was performed during the early follicular phase of two subsequent menstrual cycles (days 46). to avoid interactions between the study procedures, the study was performed at intervals of at least 2 days in men. following a 10-h overnight fast, a short polyethylene catheter was inserted into an antecubital vein for infusion of test substances at 0800 h. another catheter was placed into the contralateral forearm vein for blood sampling. a 0.9% saline infusion plus heparin (0.4 units kg min) or a lipid infusion (abbolipid 20% , abbott, wiesbaden, germany ; or lipovens 20% , fresenius kabi, bad homburg, germany) plus heparin (0.4 units kg min) was infused at a constant rate of 1.5 ml / min for 330 min. four hours after the start of the saline / heparin infusion a hyperinsulinemic - euglycemic clamp was started in six subjects. each subject was studied before and after an 8-week rosiglitazone treatment (2 8 mg / day ; glaxosmithkline, mnchen, germany) using an oral glucose tolerance test and a hyperinsulinemic - euglycemic clamp as described previously. in brief, 40 miu / m per min human insulin (actrapid ; novo nordisk, bagsvaard, denmark) and a variable infusion of 10% glucose (serag wiessner, naila, germany) was used, and capillary glucose concentration was monitored every 5 min and was maintained between 4.0 and 4.9 insulin sensitivity was assessed as m value and was calculated by dividing the average glucose infusion rate (mg glucose / min) during the steady state of the clamp by the body weight. plasma fgf-21 levels were measured before and during lhi shi, and steady state at the end of the hyperinsulinemic - euglycemic clamp. metabolic parameters, including ffas, triacylglycerols, total cholesterol, ldl cholesterol, hdl cholesterol, glucose, and insulin were determined as described previously. serum fgf-21 levels were determined by radioimmunoassay (phoenix europe) using 125 i labeled fgf-21 as tracer. the linear range of the assay was 0.58.5 ng / ml, and the standard range was 0.23430 ng / ml. the interassay and intra - assay coefficients of variation were 5.0 and 14%, respectively. all statistical procedures were performed using spss version 15.0 (spss, chicago, il). profiles of ffas, insulin, glucose, triacylglycerols, and fgf-21 were compared by repeated - measures anova with treatment and time as within - subject factors and the huynh - feldt- procedure as correction factor, which corrects for a positive biased f test for within - subject factors. the area under the concentration time curve (auc) was calculated by using the trapezoidal integration. the change in fgf-21, ffas, glucose, triacylglycerols and insulin between baseline and after lhi and shi was calculated by subtracting the baseline value from the value 4 h after each infusion procedure. data were compared by paired student's t test for normally distributed data and wilcoxon test for skewed data. the correlations between the values were estimated by pearson's correlation test and adjusted for age, sex bmi, and change in insulin. the unpaired t test or mann - whitney test was used for group comparison, as appropriate. were considered to be significant if the two - sided was < 0.05. incubation of hepg2 cells with a mixture of palmitic, linoleic, or oleic acid induced an increase in fgf expression and secretion compared with the bsa control. the analysis of the individual fatty acids revealed that this increase was not detectable for palmitic acid, while stimulation with oleic or linoleic acid ed in a significant elevation of fgf-21 secretion. within those experiments, d ), and we analyzed time course dependent changes in mrna expression (fig . the maximum of those effects was observed at 24 h for fgf-21 expression and 48 h for protein secretion, a time course in line with our in vivo experiments . furthermore, the dose - response experiments using different oleate - to - bsa ratios ( control vs. 1:1, 2:1, 4:1, 8:1, and 16:1) suggested an increase in fgf-21 secretion in a dose - dependent manner (6.61 0.81 vs. 6.93 1.25, 7.36 1.18, 8.98 1.18, 9.66 1.65, and 10.47 1.10 ng / ml ; p < 0.05 for control vs. 4:1, 8:1, and 16:1 ; p < 0.05 for 1:1 vs. 8:1 and 16:1 ; and p < 0.05 for 2:1 vs. 16:1). protein levels of fgf-21 in the supernatant of hepg2 cells after stimulation with palmitate (a), oleate (b), linoleate (c), and an ffa mixture (d) for 1, 2, 4, 8, and 24 h. are expressed as means se. * p < 0.05 vs. bsa at the same time point, p = 0.072 for fgf-21 protein levels during linoleate stimulation at 2 h vs. bsa, p = 0.069 for fgf-21 protein levels during linoleate stimulation at 8 h vs. bsa, respectively. mrna expression of fgf-21 in hepg2 cells after stimulation with linoleate for 1, 2, 4, and 8 h. are expressed as means se. * p < 0.05 vs. bsa at the same time point. to evaluate whether these effects were mediated via ppar the ppar-specific sirna induced a knockdown of ppar of 60%, although the expression of the ppar target gene cpt1a was reduced by only 35%. despite the incomplete knockdown of ppar, the increase in fgf-21 secretion and mrna expression during stimulation with linoleate and ffa mixture was not further observed. thus, at mrna and protein levels indicate that the ffa - induced effects on fgf-21 depend on activation of ppar (fig . 3). a: gene expression of fgf-21, cpt1a, and ppar in hepg2 cells in bsa medium after sirna knockdown of ppar compared with control sirna (100%). are expressed as means se. * p < 0.05 vs. expression during control sirna, respectively. b: fgf-21 mrna expression in hepg2 cells during linoleate or ffa mixture stimulation after sirna knockdown of ppar (compared with control sirna and bsa stimulation). are expressed as means se. * p < 0.05 vs. expression during control sirna, respectively. c: concentration of fgf-21 in the supernatant of hepg2 cells after sirna knockdown of ppar and subsequent stimulation by linoleate or ffa mixture compared with control sirna and bsa medium. * p < 0.05 vs. control sirna and bsa medium. as lipid infusion contains a mixture of different fatty acids, glycerol, and lecithin, we analyzed the effect of lecithin and glycerol on fgf-21 secretion in hepg2 cells. compared with controls, no significant effect of those substances was detectable in hepg2 cells. to evaluate the regulation of fgf-21 expression in human muscle actually, no fgf-21 expression was detectable in these experiments, despite accurate positive controls in hepg2 cells. those data suggest that the primary human myoblasts used here do not express fgf-21; therefore, we did not analyze the effects of ffas in those cells. there was no baseline difference of ffas before lhi and shi (p = ns). as expected, ffa levels increased during lhi, whereas only a small increase was seen during shi, ing in significantly higher ffa levels during lhi compared with shi (auc lhi vs. shi : 1,183.5 66.7 vs. 228.8 17.9 nmol p < 0.001 ; treatment - vs .- time interaction : p < 0.001) (fig . 4a). accordingly, triglyceride levels rose during lhi (p < 0.05), while a moderate decrease was observed during shi (p < 0.05), finally also ing in significantly higher triglyceride levels during lhi (auc lhi vs. shi : 1,360.6 172.0 vs. 134.0 12.5 nmol p < 0.05, p < 0.01 ; treatment - vs .- time interaction : p < 0.001) (fig . concentrations of ffas ( a), triacylglycerols (b), and fgf-21 (c) during shi and lhi . are expressed as means se. * * p < 0.01; * p < 0.05. glucose levels were unchanged during both lhi and shi (baseline : 4.91 0.80 vs. 4.84 0.54 mmol / l, treatment - vs .- time interaction : p = ns ; auc lhi vs. shi : 1,130.0 24.9 vs. 1,110.1 17.6 mmol no change of insulin levels was observed during lhi compared with baseline ( baseline : 5.17 0.53 vs. 4 h : 4.97 0.62 mu / l ; p = ns), while a small, but progressive, decline in insulin concentration was detected during shi (baseline : 5.80 0.76 vs. 4 h : 2.98 0.32 mu this decline ed in lower insulin values during shi compared with lhi ( auc lhi vs. shi : 1,384.2 132.5 vs. 1,021.8 109.8 mu p < 0.001 ; treatment - vs .- time interaction : p < 0.001). while no change of fgf-21 levels was detected during shi (p = ns), an increase in fgf-21 levels thus, fgf-21 levels were significantly higher during lhi compared with shi (treatment - vs .- time interaction : p < 0.05 . ; auc : 1,191 91 vs. 1,090 86 ng ml min ; p < 0.05) (fig . notably, we found at 300 min a progressive and highly significant increase of fgf-21 in the remaining 15 participants, who had an isolated lipid infusion over a total of 300 min ( p < 0.001 ; data not shown) interestingly, the change of fgf-21 levels compared with baseline was positively correlated to the change of ffa levels during both lhi and shi (r = 0.528, p < 0.001) (fig . 5). given the circadian changes in the control group, we also included the saline group in the correlation analysis between changes of ffas and fgf-21. most importantly, no correlation was found between the change of fgf-21 levels and the change of insulin levels during lhi and shi, while the correlation between ffas and fgf-21 was robust, even after adjustment for additional confounders (age, sex bmi, and insulin) (r = 0.474, p < 0.005). correlation between changes of fgf-21 and ffas during lhi and shi. the saline group and the lipid group were included in the correlation analysis. correlation after adjustment for sex, age, bmi, and change in insulin levels (r = 0.474, p < 0.005). as in some participants, the lipid infusion induced a reduction in fgf-21 concentrations, and we separated those participants according to the change in fgf-21 levels during lhi. however, no significant difference was found between both groups concerning their sex, age bmi whr, baseline levels of blood glucose, fasting fgf-21, fasting ffas, and homeostasis model assessment values. to delineate a potential interaction of lipid- and insulin - induced effects on fgf-21, we additionally analyzed hyperinsulinemic - euglycemic clamps, which were performed at baseline in a rosiglitazone treatment trial in 17 subjects with impaired glucose tolerance. steady - state blood samples were taken not less than 3 h after start of insulin infusion. an 700% increase of insulin levels was observed during this protocol (p < 0.005). under those conditions, a small but significant increase in fgf-21 levels during hyperinsulinemia was observed in these subjects (fig . are expressed as means se . as expected and described in numerous studies, the glucose response during the oral glucose tolerance test and insulin sensitivity . effect of ppar stimulation by rosiglitazone treatment on fgf-21 levels in subjects with impaired glucose tolerance . the accuracy of the fgf-21 assay was investigated by adding various amounts of lipid solution ( lipovens 20% ; fresenius kabi, bad homburg, germany) (0, 0.5, 1.5, and 2.5% vol / vol) to human plasma samples. this ex vivo addition of lipid infusion ed in measured triglyceride concentrations of 1.28, 3.56, 8.09, and 12.69 mmol / l. we next added 6 mmol / l oleic acid or linoleic acid to these 0, 0.5, 1.5, and 2.5% vol / vol triglyceride dilutions, ing in ffa concentrations comparable with those in plasma samples during lipid infusion (4.577.31 the subsequently measured fgf-21 values were not significantly different, neither for oleic acid nor for linoleic acid . taken together, neither triglyceride nor ffa concentrations similar to those observed in vivo in the present study interfered with the measured concentrations of fgf-21 . incubation of hepg2 cells with a mixture of palmitic, linoleic, or oleic acid induced an increase in fgf expression and secretion compared with the bsa control . the analysis of the individual fatty acids revealed that this increase was not detectable for palmitic acid, while stimulation with oleic or linoleic acid ed in a significant elevation of fgf-21 secretion . within those experiments, d), and we analyzed time course dependent changes in mrna expression (fig . the maximum of those effects was observed at 24 h for fgf-21 expression and 48 h for protein secretion, a time course in line with our in vivo experiments . furthermore, the dose - response experiments using different oleate - to - bsa ratios ( control vs. 1:1, 2:1, 4:1, 8:1, and 16:1) suggested an increase in fgf-21 secretion in a dose - dependent manner (6.61 0.81 vs. 6.93 1.25, 7.36 1.18, 8.98 1.18, 9.66 1.65, and 10.47 1.10 ng / ml ; p < 0.05 for control vs. 4:1, 8:1, and 16:1 ; p < 0.05 for 1:1 vs. 8:1 and 16:1 ; and p < 0.05 for 2:1 vs. 16:1). protein levels of fgf-21 in the supernatant of hepg2 cells after stimulation with palmitate (a), oleate (b), linoleate (c), and an ffa mixture (d) for 1, 2, 4, 8, and 24 h. are expressed as means se. * p < 0.05 vs. bsa at the same time point, p = 0.072 for fgf-21 protein levels during linoleate stimulation at 2 h vs. bsa, p = 0.069 for fgf-21 protein levels during linoleate stimulation at 8 h vs. bsa, respectively. mrna expression of fgf-21 in hepg2 cells after stimulation with linoleate for 1, 2, 4, and 8 h. are expressed as means se. * p < 0.05 vs. bsa at the same time point. to evaluate whether these effects were mediated via ppar the ppar-specific sirna induced a knockdown of ppar of 60%, although the expression of the ppar target gene cpt1a was reduced by only 35%. despite the incomplete knockdown of ppar, the increase in fgf-21 secretion and mrna expression during stimulation with linoleate and ffa mixture was not further observed. thus, at mrna and protein levels indicate that the ffa - induced effects on fgf-21 depend on activation of ppar (fig . 3). a: gene expression of fgf-21, cpt1a, and ppar in hepg2 cells in bsa medium after sirna knockdown of ppar compared with control sirna (100%). are expressed as means se. * p < 0.05 vs. expression during control sirna, respectively. b: fgf-21 mrna expression in hepg2 cells during linoleate or ffa mixture stimulation after sirna knockdown of ppar (compared with control sirna and bsa stimulation). are expressed as means se. * p < 0.05 vs. expression during control sirna, respectively. c: concentration of fgf-21 in the supernatant of hepg2 cells after sirna knockdown of ppar and subsequent stimulation by linoleate or ffa mixture compared with control sirna and bsa medium. * p < 0.05 vs. control sirna and bsa medium. as lipid infusion contains a mixture of different fatty acids, glycerol, and lecithin, we analyzed the effect of lecithin and glycerol on fgf-21 secretion in hepg2 cells. compared with controls, no significant effect of those substances was detectable in hepg2 cells. to evaluate the regulation of fgf-21 expression in human muscle actually, no fgf-21 expression was detectable in these experiments, despite accurate positive controls in hepg2 cells. those data suggest that the primary human myoblasts used here do not express fgf-21; therefore, we did not analyze the effects of ffas in those cells. there was no baseline difference of ffas before lhi and shi (p = ns). as expected, ffa levels increased during lhi, whereas only a small increase was seen during shi, ing in significantly higher ffa levels during lhi compared with shi (auc lhi vs. shi : 1,183.5 66.7 vs. 228.8 17.9 nmol p < 0.001 ; treatment - vs .- time interaction : p < 0.001) (fig . 4a). accordingly, triglyceride levels rose during lhi (p < 0.05), while a moderate decrease was observed during shi (p < 0.05), finally also ing in significantly higher triglyceride levels during lhi (auc lhi vs. shi : 1,360.6 172.0 vs. 134.0 12.5 nmol p < 0.05, p < 0.01 ; treatment - vs .- time interaction : p < 0.001) (fig . concentrations of ffas ( a), triacylglycerols (b), and fgf-21 (c) during shi and lhi . are expressed as means se. * * p < 0.01; * p < 0.05. glucose levels were unchanged during both lhi and shi (baseline : 4.91 0.80 vs. 4.84 0.54 mmol / l, treatment - vs .- time interaction : p = ns ; auc lhi vs. shi : 1,130.0 24.9 vs. 1,110.1 17.6 mmol no change of insulin levels was observed during lhi compared with baseline ( baseline : 5.17 0.53 vs. 4 h : 4.97 0.62 mu / l ; p = ns), while a small, but progressive, decline in insulin concentration was detected during shi (baseline : 5.80 0.76 vs. 4 h : 2.98 0.32 this decline ed in lower insulin values during shi compared with lhi ( auc lhi vs. shi : 1,384.2 132.5 vs. 1,021.8 109.8 mu p < 0.001 ; treatment - vs .- time interaction : p < 0.001). while no change of fgf-21 levels was detected during shi (p = ns), an increase in fgf-21 levels was found during lhi (p < 0.05). thus, fgf-21 levels were significantly higher during lhi compared with shi (treatment - vs .- time interaction : p < 0.05 . ; auc : 1,191 91 vs. 1,090 86 ng ml min ; p < 0.05) (fig . notably, we found at 300 min a progressive and highly significant increase of fgf-21 in the remaining 15 participants, who had an isolated lipid infusion over a total of 300 min ( p < 0.001 ; data not shown). interestingly, the change of fgf-21 levels compared with baseline was positively correlated to the change of ffa levels during both lhi and shi (r = 0.528, p < 0.001) (fig . 5). given the circadian changes in the control group, we also included the saline group in the correlation analysis between changes of ffas and fgf-21. most importantly, no correlation was found between the change of fgf-21 levels and the change of insulin levels during lhi and shi, while the correlation between ffas and fgf-21 was robust, even after adjustment for additional confounders (age, sex bmi, and insulin) (r = 0.474, p < 0.005). the saline group and the lipid group were included in the correlation analysis. correlation after adjustment for sex, age, bmi, and change in insulin levels (r = 0.474, p < 0.005). as in some participants , the lipid infusion induced a reduction in fgf-21 concentrations, and we separated those participants according to the change in fgf-21 levels during lhi. however, no significant difference was found between both groups concerning their sex, age bmi whr, baseline levels of blood glucose, fasting fgf-21, fasting ffas, and homeostasis model assessment values. to delineate a potential interaction of lipid- and insulin - induced effects on fgf-21, we additionally analyzed hyperinsulinemic - euglycemic clamps, which were performed at baseline in a rosiglitazone treatment trial in 17 subjects with impaired glucose tolerance. steady - state blood samples were taken not less than 3 h after start of insulin infusion. an 700% increase of insulin levels was observed during this protocol (p < 0.005). under those conditions, a small but significant increase in fgf-21 levels during hyperinsulinemia was observed in these subjects (fig . as expected and described in numerous studies, the glucose response during the oral glucose tolerance test and insulin sensitivity were improved after 8 weeks of rosiglitazone treatment . effect of ppar stimulation by rosiglitazone treatment on fgf-21 levels in subjects with impaired glucose tolerance . the accuracy of the fgf-21 assay was investigated by adding various amounts of lipid solution ( lipovens 20% ; fresenius kabi, bad homburg, germany) (0, 0.5, 1.5, and 2.5% vol / vol) to human plasma samples. this ex vivo addition of lipid infusion ed in measured triglyceride concentrations of 1.28, 3.56, 8.09, and 12.69 mmol / l. we next added 6 mmol / l oleic acid or linoleic acid to these 0, 0.5, 1.5, and 2.5% vol / vol triglyceride dilutions, ing in ffa concentrations comparable with those in plasma samples during lipid infusion (4.577.31 the subsequently measured fgf-21 values were not significantly different, neither for oleic acid nor for linoleic acid . taken together, neither triglyceride nor ffa concentrations similar to those observed in vivo in the present study interfered with the measured concentrations of fgf-21 . various recent cross - sectional studies described a positive correlation between fgf-21 levels and parameters of lipid metabolism, specifically ffas and triacylglycerols. however, existing experimental data regarding fgf-21 did not yet elucidate the direction of that relationship. both, the experimental data presented here and the of the controlled, randomized cross - over human trial strongly suggest a direct regulation of circulating fgf-21 levels in humans by ffas. interestingly, insulin appears to have an independent effect on circulating fgf-21, although our data, respective of that effect, are not based on a controlled trial and future studies are required to further strengthen that finding. in terms of physiology , the ffa - induced increase of fgf-21 might contribute to the adaption of the organism to starvation. in response to fasting , fgf-21 is known to be stimulated by activation of the ppar, a nuclear receptor, which is itself activated by fatty acids. ffas usually increase during fasting, suggesting that a stimulation of fgf-21 secretion may be induced by increased ffas directly via ppar activation, an assumption that is strongly underlined by our data. the relevance of ppar in the regulation of fgf-21 was supported by recent data showing increased fgf-21 levels after pharmacological ppar activation using fibrates. fgf-21 increased insulin sensitivity in animal experiments and attenuated the hormone - stimulated lipolysis in human adipocytes probably due to reduced expression of perilipin, a phosphoprotein that is thought to recruit several lipases to the surface of the lipid droplets for subsequent triglyceride hydrolysis. these biological properties suggest that fgf-21 may contribute to the anabolic switch of the organism under conditions of starvation and the mechanism presented here might explain the previously described elevated fgf-21 levels in patients with prevalent lipid disorders, obesity, or diabetes. the insulin - sensitizing effect of fgf-21 has been convincingly demonstrated within animal experiments. as current data suggest differential effects of saturated and unsaturated fatty acids, and especially polyunsaturated fatty acids on glucose metabolism, it is tempting to speculate that some of these effects might be mediated by fgf-21. indeed unsaturated fatty acids, and specifically the polyunsaturated linoleic acid, stimulated fgf-21 secretion, while the saturated fatty acid palmitic had no effect on fgf-21 secretion in hepg2 cells. various studies suggested that the ffa - induced activation of ppar depends on the degree of saturation of those ffas. therefore, the different effects on fgf-21 secretion may be caused by different ppar binding of saturated and unsaturated fatty acids. specific effects regarding the development of metabolic disorders remains to be elucidated. a negative correlation between fasting insulin and fgf-21 levels was recently described in diabetic subjects, while this association was reported to be positive in obese individuals. since we observed slightly higher insulin levels during lhi compared with shi in our participants, hyperinsulinemic - euglycemic clamps were performed for an in - depth analysis of the effects of insulin on fgf-21 levels. the slight increase of fgf-21 by hyperinsulinemia supports an independent effect of insulin on the regulation of fgf-21. given a well - known reduction of ffas during hyperinsulinemia, this effect may have been even underestimated. , showing that activation of akt contributes to the expression of fgf-21. thus, hyperinsulinemia and elevated levels of ffas might contribute together to the elevated fgf-21 levels seen in diabetic patients. nevertheless, both in vivo and in vitro data support a direct effect of ffas on fgf-21 expression and secretion, suggesting that the effect of ffas seen in our lipid trial would be at least in part independent of the moderate hyperinsulinemia, which occurs in the time frame presented here compared with the control group. most importantly, no correlation was found between the change of fgf-21 levels and the change of insulin levels during lhi and shi, a fact that is in line with an independent effect of ffas on fgf-21 levels. the ffa - induced effect in liver cells seems to depend on ppar stimulation, which is strongly supported by the in vitro data presented here. however, it should be mentioned in that context, other tissues like adipose tissue or muscle might also contribute to the ffa - induced increase in fgf-21 in vivo. we do not feel that the fact of absent fgf-21 expression in primary human myoblasts contradicts a substantial release of fgf-21 from muscle cells in vivo. our cells were predominantly undifferentiated cells, and the degree of differentiation might contribute to the expression pattern respective of fgf-21. a potential role of muscle cells was clearly supported by the study of izumiya et al. , although the relevance of muscle cell differentiation might require furthers studies. even if recent animal data suggested a regulation of fgf-21 also by ppar, no change of fgf-21 levels was detected during insulin - sensitizing treatment with ppar stimulation in the human trial in subjects with impaired glucose tolerance (fig . although ppar agonists may recruit different nuclear cofactors, which may explain different biological effects, our data do not support ( but also not entirely exclude) that the observed effects on fgf-21 levels during lhi were mediated in part by ppar stimulation. limitations of the present study need to be mentioned. in a recent study although small effects may have been underestimated in the abovementioned study, it is noteworthy that the increase of ffas during fasting is substantially lower in comparison to changes observed during lipid infusion. as such, although the intervention performed here is a well - established model investigating metabolic effects of hyperlipidemia, supraphysiological levels of ffas, as observed in the present study, may have reduced the period potentially required to observe effects of more moderate ffa elevations on fgf-21. in addition, ffas and triacylglycerols are both increased by lhi, and the respective effects are difficult to dissociate. although the correlation between changes in ffas and fgf-21 during lhi suggests that ffas contribute to the effect on fgf-21, we can not entirely exclude a role of triacylglycerols or other compounds within the applied infusions. notably, no effect of glycerol or lecithin was detectable in hepg2 cells, suggesting that those substances have no substancial effect on fgf-21 secretion. recent human data indicate a specific role of ffas on fgf-21 secretion by showing a positive correlation between both parameters in healthy subjects. furthermore, dostalova et al. observed not only reduced fgf-21 levels but also lower ffas in anorectic women compared with control subjects, while no difference in insulin, triacylglycerols, and fasting glucose was detected between anorectic and healthy women in this study. those data do support a model of ffas as major regulators of fgf-21 levels. in summary, we demonstrated in vitro that ffas stimulate fgf-21 expression and secretion in a human hepatic carcinoma cell line in a ppar-dependent fashion. the in vivo relevance of this finding was confirmed in a controlled, randomized cross - over trial in humans. within that trial, acutely increased levels of fgf-21 were observed. most interestingly, hyperinsulinemia also induced a small but significant increase of fgf-21, suggesting that both ffas and insulin regulate fgf-21, which may explain elevated levels of fgf-21 in obese and diabetic patients but offers also a potential mechanism linking regulation of fgf-21 to the switch of metabolism during starvation.

objectivefibroblast growth factor (fgf)-21 improves insulin sensitivity and lipid metabolism in obese or diabetic animal models, while human studies revealed increased fgf-21 levels in obesity and type 2 diabetes. given that fgf-21 has been suggested to be a peroxisome proliferator activator receptor (ppar) dependent regulator of fasting metabolism, we hypothesized that free fatty acids (ffas), natural agonists of ppar, might modify fgf-21 levels.research design and methodsthe effect of fatty acids on fgf-21 was investigated in vitro in hepg2 cells. within a randomized controlled trial, the effects of elevated ffas were studied in 21 healthy subjects (13 women and 8 men). within a clinical trial including 17 individuals, the effect of insulin was analyzed using an hyperinsulinemic - euglycemic clamp and the effect of ppar activation was studied subsequently in a rosiglitazone treatment trial over 8 weeks.oleate and linoleate increased fgf-21 expression and secretion in a ppar-dependent fashion, as demonstrated by small - interfering rna induced ppar knockdown, while palmitate had no effect. in vivo, lipid infusion induced an increase of circulating fgf-21 in humans, and a strong correlation between the change in fgf-21 levels and the change in ffas was observed. an artificial hyperinsulinemia, which was induced to delineate the potential interaction between elevated ffas and hyperinsulinemia, revealed that hyperinsulinemia also increased fgf-21 levels in vivo, while rosiglitazone treatment had no effect.the presented here offer a mechanism explaining the induction of the metabolic regulator fgf-21 in the fasting situation but also in type 2 diabetes and obesity.

at least one third of the 48 human members of the nuclear hormone receptor superfamily function as heterodimers with the retinoid x receptors, (rxrs, nr2b1 - 3). some, but not all of these heterodimers can be directly activated by rxr agonists, called rexinoids, which include the endogenous ligand 9-cis - retinoic acid (9-cis - ra) and a number of synthetic compounds. the rexinoid responsive complexes are referred to as permissive and include the peroxisome proliferator activated receptor (ppar) and the farnesoid x receptor (fxr) heterodimers; the retinoic acid receptor (rar), and thyroid hormone receptor (tr) heterodimers are non - permissive. the xenobiotic receptor car (nr1i3) is one of several rxr heterodimers that has not been assigned to either category. initial showed that car is an apparently constitutive transactivator able to activate gene expression in the absence of exogenously added ligands. the first car ligands identified, androstanol and androstenol, are inverse agonists that block this constitutive transactivation. more recently, it has been found that car mediates the characteristic response of a subset of drug metabolizing enzymes to a variety of structurally unrelated foreign compounds. these xenobiotic activators are referred to as " phenobarbital (pb) like, " based on the prototypic effects of the anti - epileptic agent on drug metabolism. the most potent activator of murine car is the high affinity agonist ligand tcpobop (1,4-bis benzene). the effects of specific rxr ligands on the car / rxr heterodimer have not been characterized, although high concentrations of both 9-cis - ra and all - trans retinoic acid have been reported to block pb induction of the car targets cyp2b1 and cyp2b2 in primary rat hepatocytes. in addition, a recent report suggests that both compounds decrease tcpobop - dependent car transactivation. in both cases, these effects were primarily attributed to sequestration of limiting amounts of rxr into ligand - dependent homodimers or heterodimers with other receptors. we have used several approaches to specifically examine the responsiveness of the car / rxr heterodimer to rexinoids. direct biochemical measurements demonstrate that rexinoids can induce coactivator binding, but can also inhibit the effects of car agonist and inverse agonist ligands. in transient transfections, however, different car / rxr heterodimer binding sites show distinct responses to rxr activation. overall, these significantly increase the potential complexity of the response of rxr heterodimers to rxr ligands and suggest a potential modulatory effect of rexinoids on xenobiotic responses. initial functional suggested that transactivation by car / rxr heterodimers is not affected by rxr ligands, including 9-cis - ra or synthetic rexinoids, indicating that car is a non - permissive rxr partner. however, gel shift studies indicated that the mobility of the car / rxr complex bound to a dr-5 element is increased by the presence of 9-cis ra, suggesting that the rxr ligand can bind to the car / rxr heterodimer. an increase in the mobility of the car / rxr complex was also observed in the presence of tcpobop or androstanol, alone or in combination with 9-cis ra (data not shown). to test the effects of rexinoids on coactivator binding in a defined biochemical system, surface plasmon resonance was used to characterize the effects of the various ligands on binding of car, rxr and car / rxr heterodimers to src-3 (also known as actr, p / cip, rac3, aib1, tram-1), an effective coactivator for car and many other members of the nuclear receptor superfamily. as expected, 9-cis - ra stimulated coactivator binding to rxr alone, and the basal interaction of car with src-3 was enhanced by tcpobop and decreased by androstanol (fig . the interaction of src-3 with the preformed and characterized car / rxr heterodimer complex was increased by either tcpobop or 9-cis - ra ( fig . strikingly, however, coactivator binding to the heterodimer complex was decreased below the basal level in the presence of both ligands . in addition, src-3 binding to the heterodimer was decreased by androstanol and this effect was largely blocked by 9-cis - ra . these effects of 9-cis - ra on coactivator binding to the preformed heterodimer contrast with the strong stimulatory effect observed with rxr alone, confirming the characterization of this complex . these demonstrate that the presence of the rxr ligand can significantly alter the response of the car / rxr heterodimer to car ligands . gst - src-3 fusion protein was immobilized on a flow cell and tested for interaction with receptors . a. overlaid of injections of apo - rxr and rxr preincubated in the presence of 9-cis ra . b. overlaid of injections of apo - car and car preincubated in the presence of tcpobop ( tc) or androstanol (an). c. overlaid of injections of preformed car / rxr heterodimers preincubated in the presence or absence of 9-cis ra and tcpobop (tc) as indicated. d. overlaid of injections of preformed car / rxr heterodimers preincubated in the presence or absence of 9-cis ra and androstanol (an) as indicated. to examine the effects of rxr ligands on car transactivation in a more functional context , the rexinoid lg1069 was combined with the car inverse agonist androstanol and the agonist tcpobop in a series of transient transfection studies using different response elements. initial studies were based on a well characterized binding site for car / rxr heterodimers, the dr-5 element from the promoter of the retinoic acid receptor 2 isoform. in agreement with the biochemical , the rexinoid blocked both the stimulatory effect of the agonist and the inhibitory effect of the inverse agonist on this element (fig . 100 nm lg1069 decreased the inhibitory effect of androstanol by as much as 70% and had an even stronger effect on tcpobop activation ( fig . consistent with previous , deletion of the helix 12 af-2 motif of car eliminated both constitutive and ligand dependent transactivation . deletion of the same motif in rxr also strongly decreased transactivation, but did not completely eliminate ligand responses ( fig . a. hepg2 cells were cotransfected with vectors expressing wild type car ( car wt), a car af-2 deletion mutant (car8) or an rxr af-2 deletion mutant (rxr19), together with a luciferase reporter containing the rare response element, in the presence or absence of androstanol, tcpobop and/or lg1069. b. a car expression vector was cotransfected into hepg2 cells with the rare - tk - luc reporter construct in the presence of solvent (open squares) or 100 nm lg1069 (solid diamonds) and increasing amounts of either androstanol or tcpobop as indicated. luciferase activity is relative to that in the absence of ligand (100%). to test whether the apparent allosteric effects of lg1069 on car transactivation vary with different response elements, the effect of the rexinoid on transactivation directed by cytochrome p450 response elements in contrast to the with the dr-5 site, but in agreement with the biochemical , lg1069 alone had a stimulatory effect on the er-6 car response element from the promoter of the cyp3a4 gene (fig . this effect was not due to rxr homodimer function, since no transactivation was observed with rxr alone . in contrast to both the dr-5 and biochemical , lg1069 did not block the stimulatory effect of tcpobop . instead, the stimulatory effect of the two agonists was retained when they were combined . combining lg1069 with the inverse agonist androstanol ed in a partial block in its inhibitory effect and an intermediate level of activity . similar were observed with the tandem dr-4 sites from the cyp2b10 gene, except that lg1069 did not have a significant stimulatory effect on its own, but did augment the effect of tcpobop . it is interesting that these two cyp elements respond somewhat differently to rxr cotransfection, which increases responses directed by the cyp3a4 element but modestly decreases responses of the cyp2b10 element ( data not shown). on both elements, similar were observed when 9-cis ra was substituted for lg1069 (data not shown). these show that the negative effects of rexinoids on tcpobop and androstanol signaling observed with the dr-5 site from the rar promoter are not observed with the dr-4 and er-6 sites from the cyp promoters. a. hepg2 cells were cotransfected with expression constructs for car and/or rxr together with a previously described luciferase construct containing a er-6 type element from the, human cyp3a4 promoter in the presence or absence of ligands. the luciferase activity presented above is relative to that directed by vector alone in the absence of ligands (100%). b. a similar transfection with a previously described reporter containing the tandem dr-4 elements from the mcyp2b10 gene. a previously described hepg2 derivative stably expressing mouse car was used to determine the effects of rxr agonists on car function in the context of native chromatin. expression of cyp2b6, a human relative of cyp2b10, is constitutively induced in this cell line. in contrast to the with the transient transfections with the cyp response elements, cyp2b6 expression was decreased to a comparable extent by androstanol, lg1069, and the combination of both ligands. cyp2b6 expression was increased by tcpobop, as expected, and this response was blocked by lg1069 treatment. initial functional suggested that transactivation by car / rxr heterodimers is not affected by rxr ligands, including 9-cis - ra or synthetic rexinoids, indicating that car is a non - permissive rxr partner. however, gel shift studies indicated that the mobility of the car / rxr complex bound to a dr-5 element is increased by the presence of 9-cis ra, suggesting that the rxr ligand can bind to the car / rxr heterodimer. an increase in the mobility of the car / rxr complex was also observed in the presence of tcpobop or androstanol, alone or in combination with 9-cis ra (data not shown). to test the effects of rexinoids on coactivator binding in a defined biochemical system, surface plasmon resonance was used to characterize the effects of the various ligands on binding of car, rxr and car / rxr heterodimers to src-3 (also known as actr, p / cip, rac3, aib1, tram-1), an effective coactivator for car and many other members of the nuclear receptor superfamily. as expected, 9-cis - ra stimulated coactivator binding to rxr alone, and the basal interaction of car with src-3 was enhanced by tcpobop and decreased by androstanol (fig . the interaction of src-3 with the preformed and characterized car / rxr heterodimer complex was increased by either tcpobop or 9-cis - ra ( fig . strikingly, however, coactivator binding to the heterodimer complex was decreased below the basal level in the presence of both ligands . in addition, src-3 binding to the heterodimer was decreased by androstanol and this effect was largely blocked by 9-cis - ra . these effects of 9-cis - ra on coactivator binding to the preformed heterodimer contrast with the strong stimulatory effect observed with rxr alone, confirming the characterization of this complex . these demonstrate that the presence of the rxr ligand can significantly alter the response of the car / rxr heterodimer to car ligands . gst - src-3 fusion protein was immobilized on a flow cell and tested for interaction with receptors . a. overlaid of injections of apo - rxr and rxr preincubated in the presence of 9-cis ra . b. overlaid of injections of apo - car and car preincubated in the presence of tcpobop ( tc) or androstanol (an). c. overlaid of injections of preformed car / rxr heterodimers preincubated in the presence or absence of 9-cis ra and tcpobop (tc) as indicated. d. overlaid of injections of preformed car / rxr heterodimers preincubated in the presence or absence of 9-cis ra and androstanol (an) as indicated. to examine the effects of rxr ligands on car transactivation in a more functional context, the rexinoid lg1069 was combined with the car inverse agonist androstanol and the agonist tcpobop in a series of transient transfection studies using different response elements. initial studies were based on a well characterized binding site for car / rxr heterodimers, the dr-5 element from the promoter of the retinoic acid receptor 2 isoform. in agreement with the biochemical , the rexinoid blocked both the stimulatory effect of the agonist and the inhibitory effect of the inverse agonist on this element (fig . 2a). in more detailed dose response studies, 100 nm lg1069 decreased the inhibitory effect of androstanol by as much as 70% and had an even stronger effect on tcpobop activation (fig . consistent with previous , deletion of the helix 12 af-2 motif of car eliminated both constitutive and ligand dependent transactivation . deletion of the same motif in rxr also strongly decreased transactivation, but did not completely eliminate ligand responses ( fig . a. hepg2 cells were cotransfected with vectors expressing wild type car ( car wt), a car af-2 deletion mutant (car8) or an rxr af-2 deletion mutant (rxr19), together with a luciferase reporter containing the rare response element, in the presence or absence of androstanol, tcpobop and/or lg1069. b. a car expression vector was cotransfected into hepg2 cells with the rare - tk - luc reporter construct in the presence of solvent (open squares) or 100 nm lg1069 (solid diamonds) and increasing amounts of either androstanol or tcpobop as indicated. luciferase activity is relative to that in the absence of ligand (100%). to test whether the apparent allosteric effects of lg1069 on car transactivation vary with different response elements, the effect of the rexinoid on transactivation directed by cytochrome p450 response elements was examined. in contrast to the with the dr-5 site, but in agreement with the biochemical , lg1069 alone had a stimulatory effect on the er-6 car response element from the promoter of the cyp3a4 gene (fig . this effect was not due to rxr homodimer function, since no transactivation was observed with rxr alone . in contrast to both the dr-5 and biochemical , lg1069 did not block the stimulatory effect of tcpobop . instead, the stimulatory effect of the two agonists was retained when they were combined . combining lg1069 with the inverse agonist androstanol ed in a partial block in its inhibitory effect and an intermediate level of activity . similar were observed with the tandem dr-4 sites from the cyp2b10 gene, except that lg1069 did not have a significant stimulatory effect on its own, but did augment the effect of tcpobop . it is interesting that these two cyp elements respond somewhat differently to rxr cotransfection, which increases responses directed by the cyp3a4 element but modestly decreases responses of the cyp2b10 element ( data not shown). on both elements, similar were observed when 9-cis ra was substituted for lg1069 (data not shown). these show that the negative effects of rexinoids on tcpobop and androstanol signaling observed with the dr-5 site from the rar promoter are not observed with the dr-4 and er-6 sites from the cyp promoters. a. hepg2 cells were cotransfected with expression constructs for car and/or rxr together with a previously described luciferase construct containing a er-6 type element from the, human cyp3a4 promoter in the presence or absence of ligands. the luciferase activity presented above is relative to that directed by vector alone in the absence of ligands (100%). b. a similar transfection with a previously described reporter containing the tandem dr-4 elements from the mcyp2b10 gene. a previously described hepg2 derivative stably expressing mouse car was used to determine the effects of rxr agonists on car function in the context of native chromatin. expression of cyp2b6, a human relative of cyp2b10, is constitutively induced in this cell line. in contrast to the with the transient transfections with the cyp response elements, cyp2b6 expression was decreased to a comparable extent by androstanol, lg1069, and the combination of both ligands. cyp2b6 expression was increased by tcpobop, as expected, and this response was blocked by lg1069 treatment. a number of rxr heterodimer complexes have been categorized as either permissive or non - permissive based on their responses to rxr ligands. although there are some reported discrepancies, the ppar, fxr and ngf - ib / nurr1 complexes are generally considered permissive, while the non - permissive group includes the rar, tr and vdr complexes (see). in the permissive complexes, coactivators can presumably bind to the af-2 surface of both partners and binding to the rxr partner is stimulated by rexinoid agonists. this can in distinct responses depending on which of the two partners is activated. in the case of the ppar/rxr complex, for example, activation with ppar or rxr specific ligands in recruitment of quite different coactivators. among the non - permissive complexes, recent suggest that the c - terminal helix 12 of rxr exerts allosteric effects on rar function. however, additional studies suggest that corepressor binding to the apo - rar component of the heterodimer blocks rexinoid induced binding of coactivators to rxr. these suggest that the mechanisms that account for the lack of responsiveness of the non - permissive complexes are complex. as summarized in fig. 5 , the described here demonstrate that the effects of rxr agonists on the car / rxr heterodimer can be quite different in different contexts. in vitro studies in the absence of dna demonstrate that car / rxr heterodimers can be activated by an agonist ligand for either receptor. this positive response to 9-cis - ra contrasts with the inhibition that would from the loss of rxr to ligand - dependent homodimers, confirming that these studies reflect the activity of car / rxr heterodimers and demonstrating that such complexes can be permissive for rxr signaling. remarkably, however, coactivator binding is decreased in the presence of both of the activating ligands. similarly, the inhibitory effect of the inverse agonist ligand androstanol on coactivator recruitment is blocked by 9-cis - ra. total rna was prepared from a previously described hepg2 derivative stably expressing mcar and cultured in the presence of the indicated ligands (an, androstanol ; lg, lg1069 ; tc, tcpobop) for 9 hours. expression of cyp2b6 and the ef1 control was analyzed by northern blotting using appropriate cdna probes and quantitated by phosphorimager analysis. very similar were obtained in 3 independent experiments; a representative experiment is shown in transient transfections, the rexinoid lg1069 shows different effects on the ability of the xenobiotic receptor to transactivate various response elements. the rexinoid alone has no effect on transactivation of the rare, but strongly inhibits responses to both the agonist tcpobop and the inverse agonist androstanol. in contrast, lg1069 can stimulate transactivation of the response element from the cyp3a4 promoter. on that element it does not block the stimulatory effect of tcpobop, but decreases the inhibitory effect of the androstanol somewhat. on the tandem cyp2b10 elements , lg1069 does not have a stimulatory effect on its own and does not decrease the inhibitory effect of androstanol, but it does increase response to tcpobop. finally, expression of the endogenous cyp2b6 gene in car expressing hepg2 cells is modestly inhibited by lg1069 alone, androstanol alone and the combination of both. in this context, the rexinoid blocks activation by the agonist tcpobop. these rexinoid responses share the general theme of inhibition of some of the effects of car ligands by rxr ligands, but are clearly quite disparate. the differences may be based on variations in experimental approach or on more fundamental functional differences. for example, it is possible that the inhibitory effect of lg1069 in the stable car expressing cell line is based on the sequestration of limiting amounts of rxr into ligand - dependent homodimers, as has been suggested in previous studies using primary hepatocytes. however, similar effects were observed with the relatively large amounts of rxr used for the biochemical studies or with the transient transfections, which relied on cotransfected rxr. it is also possible that differences in the chromatin context could contribute to the differences between the effects observed with the endogenous and transiently transfected promoters. however, such differences can not account for the with reporter plasmids that differ only in the identity of the response element. overall, we conclude that binding of both the dna and the rxr ligands can in allosteric effects on the function of the car / rxr heterodimer. the disparities in these responses preclude a single, simple explanation for the effects of rexinoids on car / rxr heterodimers. nonetheless, one attractive possibility is that binding of the rxr agonist exerts direct inhibitory effects on either the binding or functional effects of the car ligands. some precedence for a significant allosteric effect of one heterodimer partner on another is provided by the inability of the insect ecdysone receptor to bind its steroid ligand in the absence of its heterodimer partner usp. the outcome of this inhibition of car ligand binding would be somewhat different depending on whether or not the rxr agonist was able to activate the basal transactivation of car / rxr heterodimer bound to a particular element. on the rare, for example, the rxr agonist might bind, but not increase transactivation beyond that observed in the absence of ligands. in this case, blocking tcpobop binding in the case of the cyp3a4 response element, however, activation by the rxr agonist is comparable to that observed with tcpobop alone. thus, blocking tcpobop binding would not in the net inhibitory effect observed with the rare, but could account for the lack of additive effect of the two ligands. a variety of factors complicate this relatively simple model, including the specific cytoplasmic to nuclear translocation that is required for car transactivation in the liver. rexinoids by themselves are apparently unable to induce this translocation process in rat or mouse (data not shown) hepatocytes and thus can not directly activate car targets. more complex combinatorial effects of other coactivators or corepressors (e.g.) could also contribute to the observed responses. recent suggest that car can interact with ncor or smrt in the presence of inverse agonists, although such interactions appear much weaker than those of other receptors. further characterization of potential car cofactors, as well as the development of sensitive and direct ligand binding assays will be required to define the molecular mechanisms that account for the diverse effects of rexinoids on car transactivation. overall, we conclude that the disparate effects of rexinoids on car / rxr heterodimers in different contexts add significantly to the potential complexity of the effects of rxr ligands on such nuclear receptor heterodimers, and demonstrate that classifying a particular complex as permissive or non - permissive can be an oversimplification. the described here also suggest that rexinoids may have modulatory effects on xenobiotic responses, and that such effects may differ for different car / rxr target genes. a systematic examination of the effects of rxr ligands and retinoid status on a number of the growing set of car target genes the effect of ligands on coactivator binding was evaluated using the biacore instrument as described. for interaction of car, rxr and car / rxr heterodimer with src-3, a gst - src-3 fusion protein containing the receptor interaction domain of the coactivator was expressed in bacteria and purified. lack of binding of free gst demonstrated that the surface was saturated with gst - src3. purified car and rxr proteins were produced in bacteria and preincubated overnight at 4c alone or in combination, and in the presence or absence of 10 m androstanol, 10 m tcpobop, 10 m of 9 cis - ra, as appropriate. car alone, rxr alone, and car / rxr heterodimers bound to various ligands were injected individually into flow cells and binding was measured as described. the rare - tk - luc reporter plasmid with three copies of the dr-5 motif from the promoter of the mouse rar2 gene has been described previously, as has the cyp2b10-tk - luc reporter with two copies of the dr-4 type elements from the promoter of the mouse cyp2b10 gene and the cyp3a4-tk - luc reporter with three copies of the er-6 element from the human cyp3a4 promoter. the cdm8-mcar8 deletion construct containing a deletion of the last 8 amino acids and the mutant with 2 helix 3 mutations (f171l, i174a) that block ligand binding and response have been described previously, as has the cdm8-rxr19 construct with a deletion of the last 19 amino acids. hepg2 cells were maintained in dulbecco's modified eagle medium (dmem) supplemented with 10% fetal bovine serum (hyclone). for transfections, 10hepg2 cells were plated in 24 well dishes with dmem supplemented with 10% charcoal stripped serum and transfected using the calcium phosphate precipitation method as described. the next morning typically, transfections included 100200 ng of luciferase reporter plasmids, 100 ng of -galactosidase (-gal) internal control plasmid (cmv -gal), and 100 ng of cdm8 expression vectors. cells were assayed for luciferase (promega) activities 24 hours after addition of ligands and reporter expression was normalized to -galactosidase activity (tropix) according to the manufacturer's directions. a previously described derivative of hepg2 cells stably expressing mcar was used for rna analysis. cells were subcultured and incubated in dmem supplemented with 10% charcoal stripped bovine serum overnight. various ligands were added and total rna was prepared 9 hours later and used for northern blotting using standard procedures. the effect of ligands on coactivator binding was evaluated using the biacore instrument as described. for interaction of car, rxr and car / rxr heterodimer with src-3, a gst - src-3 fusion protein containing the receptor interaction domain of the coactivator was expressed in bacteria and purified. lack of binding of free gst demonstrated that the surface was saturated with gst - src3. purified car and rxr proteins were produced in bacteria and preincubated overnight at 4c alone or in combination, and in the presence or absence of 10 m androstanol, 10 m tcpobop, 10 m of 9 cis - ra, as appropriate. car alone, rxr alone, and car / rxr heterodimers bound to various ligands were injected individually into flow cells and binding was measured as described. the rare - tk - luc reporter plasmid with three copies of the dr-5 motif from the promoter of the mouse rar2 gene has been described previously, as has the cyp2b10-tk - luc reporter with two copies of the dr-4 type elements from the promoter of the mouse cyp2b10 gene and the cyp3a4-tk - luc reporter with three copies of the er-6 element from the human cyp3a4 promoter. the cdm8-mcar8 deletion construct containing a deletion of the last 8 amino acids and the mutant with 2 helix 3 mutations (f171l, i174a) that block ligand binding and response have been described previously, as has the cdm8-rxr19 construct with a deletion of the last 19 amino acids. hepg2 cells were maintained in dulbecco's modified eagle medium (dmem) supplemented with 10% fetal bovine serum (hyclone). for transfections, 10hepg2 cells were plated in 24 well dishes with dmem supplemented with 10% charcoal stripped serum and transfected using the calcium phosphate precipitation method as described. the next morning typically, transfections included 100200 ng of luciferase reporter plasmids, 100 ng of -galactosidase (-gal) internal control plasmid (cmv -gal), and 100 ng of cdm8 expression vectors. cells were assayed for luciferase (promega) activities 24 hours after addition of ligands and reporter expression was normalized to -galactosidase activity (tropix) according to the manufacturer's directions. a previously described derivative of hepg2 cells stably expressing mcar was used for rna analysis. cells were subcultured and incubated in dmem supplemented with 10% charcoal stripped bovine serum overnight. various ligands were added and total rna was prepared 9 hours later and used for northern blotting using standard procedures. i. t. and s. s. c. carried out the studies of car function in transiently transfected cells. s. s. c. carried out the studies of gene expression in stable car expressing cells. the rightward bar (----|) symbolizes a blockage of the effect of the car ligand by rexinoids (or decrease in the case of the repression of the cyp elements by androstanol); (-), no effect.

car / rxr heterodimers bind a variety of hormone response elements and activate transcription in the absence of added ligands. this constitutive activity of murine car can be inhibited by the inverse agonist ligand androstanol or increased by the agonist tcpobop. rxr agonists activate some rxr heterodimer complexes, which are termed permissive, while other non - permissive complexes are not responsive to such ligands.direct protein - protein interaction studies demonstrate that the rxr agonist 9-cis - ra increases interaction of car / rxr heterodimers with the coactivator src-3, but also inhibits the ability of tcpobop to increase and androstanol to decrease coactivator binding. car transactivation of a response element with a five nucleotide spacer (dr-5) is unaffected by 9-cis - ra or the synthetic rxr agonist lg1069. in agreement with the inhibitory effect observed in vitro, these rexinoids block both the tcpobop mediated transactivation of this element and the androstanol dependent inhibition. in contrast , car transactivation of other response elements is increased by rexinoids. stable expression of car in a hepg2 derived cell line increases expression of the endogenous car target cyp2b6. this expression is further increased by tcpobop but decreased by either androstanol or lg1069, and lg1069 blocks the stimulatory effect of tcpobop but not the inhibitory effect of androstanol.we conclude that car / rxr heterodimers are neither strictly permissive nor non - permissive for rxr signaling. instead, rexinoids have distinct effects in different contexts. these expand the potential regulatory mechanisms of rexinoids and suggest that such compounds may have complex and variable effects on xenobiotic responses.

chronic bullous dermatosis of childhood presents in prepubertal, often preschool, children and rarely in infancy. vesicles or bullae develop on an erythematous or normal base, occasionally giving rise to a so - called string of pearls, a characteristic lesion in which peripheral vesicles develop on a polycyclic plaque. they involve the buttocks, lower abdomen and genitalia and characteristically have a perioral distribution on the face. the disorder usually remits by the age of 6 - 8 years, but in one series, only 12% of the patients experienced persistent disease. in this case report, we present an unusual example of chronic bullous dermatosis of childhood, characterized by the presence of a subepidermal cell poor blister, fullhouse deposition of immunoglobulins including prominent deposition of iga at the dermo - epidermal junction in the absence of other autoimmune and connective tissue disorders. a 14-month - old girl from saudi was admitted to the dermatology ward because of a 2-month history of skin blistering with severe itching. on examination, multiple vesiculated and crusted skin lesions were found on the extremities, face, mouth, and genitalia. no skin lesions on trunk or oral mucosa were noted. also, there was no history of drug intake, fever, or other systemic symptoms. a clinical differential diagnosis of chronic bullous disease of childhood and epidermolysis bullosa was made, and biopsies were taken from lesional and prelesional skin for routine histopathological and immunofluorescence studies, respectively. (a) chronic bullous disease of childhood. (b) chronic bullous disease of childhood. eroded and vesicular skin lesions on the external genitalia histopathological examination of a punch biopsy taken from lesional skin showed a cell - poor subepidermal blister containing scanty lymphocytes and some fibrin strands. the papillary dermis showed evidence of festooning with scanty perivascular mononuclear inflammatory cells infiltration. there was no evidence of an increase in polymorphous leukocytes or eosinophils. direct immunofluorescence studies were performed and showed strong linear deposition of iga, igg, and c3 at the dermoepidermal junction with similar but moderately strong deposition of igm (2 +) in the same region. the c3 deposits extended in a linear fashion to the skin appendages. according to the patient age, clinical distribution of the lesions along with the histological and immunofluorescence findings, a differential diagnosis was made of chronic bullous dermatosis of childhood with an unusual immunofluorescence pattern and a childhood onset of epidermolysis bullosa acquisita (eba). direct immunofluorescence study showing strong linear deposits of iga at the dermo - epidermal / basement membrane zone. (direct if 200) based on this differential diagnosis, we performed a salt split test, which showed deposition of igg and c3 at the roof of blister. these findings led to a final diagnosis of chronic bullous dermatosis of childhood (linear iga bullous dermatosis), with an unusual immunofluorescence pattern that is rarely described in the literature. histopathological examination of a punch biopsy taken from lesional skin showed a cell - poor subepidermal blister containing scanty lymphocytes and some fibrin strands. the papillary dermis showed evidence of festooning with scanty perivascular mononuclear inflammatory cells infiltration. there was no evidence of an increase in polymorphous leukocytes or eosinophils. direct immunofluorescence studies were performed and showed strong linear deposition of iga, igg, and c3 at the dermoepidermal junction with similar but moderately strong deposition of igm (2 +) in the same region. the c3 deposits extended in a linear fashion to the skin appendages. according to the patient age, clinical distribution of the lesions along with the histological and immunofluorescence findings, a differential diagnosis was made of chronic bullous dermatosis of childhood with an unusual immunofluorescence pattern and a childhood onset of epidermolysis bullosa acquisita (eba). direct immunofluorescence study showing strong linear deposits of iga at the dermo - epidermal / basement membrane zone. (direct if 200) based on this differential diagnosis, we performed a salt split test, which showed deposition of igg and c3 at the roof of blister. these findings led to a final diagnosis of chronic bullous dermatosis of childhood (linear iga bullous dermatosis), with an unusual immunofluorescence pattern that is rarely described in the literature. histopathological examination of a punch biopsy taken from lesional skin showed a cell - poor subepidermal blister containing scanty lymphocytes and some fibrin strands. the papillary dermis showed evidence of festooning with scanty perivascular mononuclear inflammatory cells infiltration. there was no evidence of an increase in polymorphous leukocytes or eosinophils. direct immunofluorescence studies were performed and showed strong linear deposition of iga, igg, and c3 at the dermoepidermal junction with similar but moderately strong deposition of igm (2 +) in the same region. the c3 deposits extended in a linear fashion to the skin appendages. according to the patient age, clinical distribution of the lesions along with the histological and immunofluorescence findings, a differential diagnosis was made of chronic bullous dermatosis of childhood with an unusual immunofluorescence pattern and a childhood onset of epidermolysis bullosa acquisita (eba). direct immunofluorescence study showing strong linear deposits of iga at the dermo - epidermal / basement membrane zone. (direct if 200) based on this differential diagnosis, we performed a salt split test, which showed deposition of igg and c3 at the roof of blister. these findings led to a final diagnosis of chronic bullous dermatosis of childhood (linear iga bullous dermatosis), with an unusual immunofluorescence pattern that is rarely described in the literature. chronic bullous dermatosis of childhood is the most common chronic bullous dermatosis in the first 10 years of life. the disease commonly involve the perioral skin, lower trunk, inner thighs, genitalia and perineum. the mucous membranes may also be affected. bullae are the major clinical finding, and their appearance is characteristic with an annular arrangement of sausage - shaped bullae around resolving crusted lesions. the disorder is self - limiting and clears up within several months to three years. the clinical and histopathological differential diagnosis usually includes dermatitis herpetiformis, childhood form of eba and bullous pemphigoid. in our case, however, the presence of strong linear deposition of iga at the dermoepidermal junction and the of the salt split test helped eliminate this possibility. furthermore, the presence of associated deposits of igg, igm, and c3, which was confirmed by immunofluorescence studies in the absence of an associated autoimmune systemic disease is a rare event in this disorder and has only partially been reported previously in the literature. powell et al. had only deposits of iga with associated igg appearing in the early stages of the illness while our case has confirmed fullhouse deposition of ig's and c3. the lack of eosinophilic infiltration, besides the presence of iga deposits excludes an early onset of bullous pemphigoid. we also believe that the cell poor appearance of the blisters in this condition could be due to the age of the illness, as suggested by the 2-month history of the eruption. chronic bullous dermatosis of childhood is a common blistering disease, which can be seen in infants and preschool children. both dermatopathologists and clinical dermatologists should be aware of the unusual and rare histopathological and immunofluorescence features, which can be seen in such cases and may lead to an erroneous diagnosis of a more severe disorder like childhood eba.

linear iga bullous dermatosis is a rare sulfone - responsive subepidermal blistering disorder of unknown etiology in which smooth linear deposits of iga are found in the basement membrane zone. chronic bullous dermatosis of childhood is equivalent to linear iga disease of adulthood and is characterized by an abrupt onset of large, widespread and tense bullae on a normal or erythematous base. in this case, we describe an unusual presentation of chronic bullous dermatosis in a 14-month - old saudi girl. histopathological examination revealed subepidermal cell poor blisters with linear deposition of iga, igg, igm, and c3 along the dermoepidermal junction. the unusual clinical, histopathological and immunofluorescence findings in this patient are discussed, with an account on the differential diagnosis in such cases along with a detailed review of the relevant literature.

human gastrointestinal (gi) tract is compartmentalized into different sections, which allows digestion and absorption of nutrients in the body. the distinct part of the gut has different physiological conditions that shape the gut microbiome (or microbial diversity within the gut). the human intestinal microbiota is composed of 10 to 10 microorganisms whose collective genome (microbiome) contains at least 100 times as many genes as our own genome. as in most mammals, the gut microbiome is dominated by four bacterial phyla namely firmicutes, bacteroidetes, actinobacteria, and proteobacteria , representing more than 1000 different molecular species or phylotypes. stomach of the gi tract has very low ph ~ 2.5, urea stress and microaerophilic environment making it extreme niche. to adopt up with this condition organism have to have some additional machinery to survive and perform its functions. there has been an estimate that human stomach comprises 32 phylotypes from proteobacteria, including 3 uncharacterized phylotypes. helicobacter pylori (h. pylori), known causative agent of peptic ulcers and gastric cancer, colonize the stomach of approximately 50% of the world's population. however now there are few recent reports indicating presence of bacteria other than h. pylori in the human stomach. our group had isolated a bacterium from non ulcer dyspeptic individual from north india in 2005 and was identified as ochrobactrum intermedium by 16s rrna sequencing and its first draft genome sequencing was reported by using ion torrent personal genome machine (pgm),. to gain insights into genomic re - architecturing in terms of gain and loss during continuous passaging for several generations and also to evaluate sequencing platform for getting more refined data (in terms of coverage), we re - sequenced the ochrobactrum intermedium m86 strain with illumina miseq and compared with 10 year earlier draft genome of the same strain. the 16s rrna based phylogenetic analysis showed ochrobactrum and brucella are closely related than helicobacter. urease gene (urec) based tree clearly indicated the presence of a distinct class of urease in ochrobactrum when compared to helicobacter and brucella (data not shown). whole - genome re - sequencing of o. intermedium m86 was conducted with the illumina miseq. we obtained 5,384,581 paired - end reads of 300 bp using illumina miseq which was 4.1 fold higher than the reads obtained from ion torrent pgm machine. sequence yield per run is higher in illumina miseq than ion torrent pgm machine and reports also showed the error rate is higher in pgm 1.71% than illumina miseq, 0.4%.the number of error - free reads, without a single mismatch or indel, was 76.45%, 15.92% for, miseq, ion torrent respectively. this comparison revealed efficiency in terms of quantity and quality miseq is somewhat better than pgm in terms of output data. the genome of passaged / re - sequenced / laboratory adapted m86 comprised of two circular chromosomes with a total size of 4,683,452 bases with a 57% g + c content and no plasmids. of the 4415 (cdss) genes predicted, 38 trna and 5 rrna genes were found (table 1). the genes were distributed into 470 subsystems using rast. to check the quality of assemblies obtained from velvet with different k - mer values; genome coverage, n50 values and maximum / median / average contig sizes were compared. it was observed that at k - mer = 25, estimated coverage was 53%, 2,642,663 2(a), (d), and (e). in case of k - mer = 57, n50 value was maximum but the coverage attained was 79%, moreover number of assembled nucleotides was less as compared to k - mer = 31 as mentioned in fig. 2(c), (d) and (e). at k - mer = 31, all the parameters were optimized and maximum coverage was obtained (fig . the de novo assembly using the velvet and spades method generated 121 contigs, which were further assembled into 34 scaffolds . sequencing statistics of earlier genome ( did on ion pgm) and resequenced strain (on illumina) was also compared. resequenced genome on illumina was better on ion pgm on all technical aspects like number of reads (5 times), genome coverage (10 times), n50 value (1.25 higher), number of scaffolds (equal) except run time (4 times more). this study was also aimed to enhance the quality of reads by re - sequencing for futuristic view of functional genomics. in our study, illumina miseq and pgm were performed to sequence genome of o. intermedium m86 and we could conclude that the illumina probably had higher coverage as compared to pgm with respect to total reads obtained and coverage, which could also be useful for larger genomes. for chromosome 1 and 2, the is finder blast showed unique insertion sequences / tandem repeats which were not reported in earlier draft genome but appeared in current analysis. unique insertion sequences that we detected only in re sequenced strain were is2, is51, is151, is407, isas1, is30, and is1595. the rast annotation system showed absence of prophage encoding genes (confirmed by phast) in earlier strain, but were present in re sequenced strain, although they were not complete indicative of a possibility of gene exchange via transduction mechanism. reference based mapping of o. intermedium m86 draft genome and resequenced genome with o. intermedium lmg3301 revealed that resequenced genome appeared to have undergone insertion as well as deletion of some genes or islands (fig . further investigation of the deleted portion belonged to phage proteins that were excised probably during passaging . anti - smash analysis revealed interesting findings with respect to absence of trps - t3pks system in the resequenced strain, with replacement of terpene and arylpolyene . such differences in biosynthetic gene cluster could assist the strains in developing and combating strategies . as was previously reported the strains of o. intermedium were resistant to all -lactam antibiotics except imipenem . this was confirmed in resequenced genome by rast annotation server and it showed the presence of -lactamase . in addition, metal - dependent hydrolases of the beta - lactamase superfamily i and beta - lactamase class c, other penicillin binding proteins, fluoroquinolones and streptothricin resistant genes were also found in resequenced as well as earlier genome but not reported so far . diverse ochrobactrum sps . have been reported from contaminated habitat which showed its role in bioremediation and tolerance to different heavy metals like arsenic, nicotine, and were found in earlier as well as resequenced strain . in addition, presence of genes coding for arsenic ( arsh, acr3, arsenate reductase) copper (cute, ccmh, copc, copd, copper chaperone, copg, ccmm, copper - translocating p - type atpase, blue copper oxidase cueo precursor), cobalt - zinc - cadmium resistance (co / zn / cd efflux system membrane fusion protein) and mercuric reductase were also detected in earlier and resequenced strain indicating the necessity of these genes for growth under in - vitro as well as in - vivo environment. strain differences under selective or non - selective pressure may lead to formation of snps leading to inter and intra - strain diversity. around 8878 snps were detected among previous genome and resequenced o. intermedium m86 (both chromosomes), wherein no snps detected in rpob, trpe, reca, dnak, aroc and gap. genes responsible for survival in acidic condition of stomach (urec, ured, uree, uref and ureg of urease operon) and flagellar assembly (flic) were devoid of snps indicating their stability under in - vitro conditions outside simulated niche. in chromosome i , we detected 2 gaps from which one fragment is absent in both the genomes, previous genome and resequenced genome. the other gap which is specific to resequenced genome includes phage related protein like phage tail protein, assembly protein. interestingly in chromosome 2, a 5 kb fragment which was absent in previous genome and also in o. intermedium lmg 3301 strain was detected in a re - sequenced strain. further, tblastx analysis of this fragment showed the presence of integrase gene from rhizobium spp. we speculate that the insertion might be of a horizontal gene transfer event in o. intermedium m86. integrase gene product mainly helps in the site specific recombination through attb and attp site. construction of vector containing attachment site and integrase gene can be use as engineering tool for insertion of desired fragment of gene through site specific recombination. it is just because crossover of nucleic acid fragment and host integration site can occur at any 3 sublocation within a trna can occur, two with flanking symmetry (anticodon - loop and t - loop tdna) and the third at the asymmetric 3 end of the gene. apart from 5 kb indel, genes absent only in resequenced genome were phage encoded protein like phage tail coding protein, dna packaging protein, hk97 family phage genes which mainly play an important role in connecting the dna filled head to tail via connector. another gene holotricin-3 precursor which is a defense response gene and chloramphenicol acetyltransferase which provides resistant to chloramphenicol antibiotic were also absent. the stability and effect of indel events on the overall physiology, regulatory and colonization mechanisms in the passaged strain needs to be investigated for acid resistance, colonization, and persistence in hostile environment of acid and urea present in stomach. considering the opportunities and challenges of niche specialized o. intermedium strain (among genus ochrobactrum) that has a tendency of genome reduction in the technology driven era. our study is significant for this isolate of gastric origin in terms of genome architecture and genome rearrangement in terms of deletion as well as insertional events during routine maintenance under laboratory conditions. given the understanding of having such property of recombination and specialized process of adaptation in diverse environment, the more understanding of ochrobactrum genus at genomic level could be important to understand if specialized set of genes are assigned for environmental and clinical adaptations. strain o. intermedium m86 was weekly passaged for about 100 generations for this study and used as laboratory adapted strain in this study. then grown in macconkey broth (himedia, india) and after overnight incubation at 37 c purity of the culture was checked and pellet was collected by centrifugation. chromosomal dna isolation was done by using pure - link dna isolation kit (invitrogen, carlsbad, usa) and quality of the dna was checked by recording absorbance at a260/280 nm and agarose gel electrophoresis. phylogenetic analysis of 16s rrna genes for o. intermedium m86, brucella melitensis biovar suis, helicobacter pylori atcc 43504 and o. intermedium lmg 3301 was done using mega v 6.0 with upgma method (bootstrap = 1000) as described. similarly urease alpha subunit (ure c) of genera ochrobactrum, helicobacter and brucella was used to build upgma tree using mega. genome re - sequencing of m86 strain was done using illumina miseq m02845 sequencer was carried out using 2 300 bp chemistry. to check the quality of paired end poor quality reads (phred quality below q30) and adapter sequences were removed using fastq trimmer and flexbar pre - processing tool respectively. to preserve mate integrity and to filter sequences by composition de novo assembly of prepossessed fastq reads was performed using velvet 1.2.10 (k - mer = 25, 31, 57) and spades 3.0.0 (k - mer 21, 33, 55). further the quality of integrated contigs / scaffolds was evaluated using check bacterial contigs tool. contigs of o. intermedium m86 draft genome and resequenced genome were mapped against chromosome i and ii of o. intermedium lmg 3301 and visualized using brig (fig . metabolic pathway prediction was performed on kaas to assign kegg orthology ( ko) numbers to each predicted cds. comparative analysis was carried out between previous draft genome and resequenced genome for different features such as presence of insertion sequences, tandem repeats, phage genes, clustered regularly interspaced short palindromic repeats (crispr), plasmid and secondary metabolite production genes using is finder, tandem repeats finder, phast, crisprfinder, blast and anti - smash 2.0 respectively. o. intermedium m86 draft genome and resequenced genome were compared with o. intermedium lmg 3301 for single nucleotide polymorphism (snps) detection using parsnp pipeline. strain o. intermedium m86 was weekly passaged for about 100 generations for this study and used as laboratory adapted strain in this study. then grown in macconkey broth (himedia, india) and after overnight incubation at 37 c purity of the culture was checked and pellet was collected by centrifugation. chromosomal dna isolation was done by using pure - link dna isolation kit (invitrogen, carlsbad, usa) and quality of the dna was checked by recording absorbance at a260/280 nm and agarose gel electrophoresis. phylogenetic analysis of 16s rrna genes for o. intermedium m86, brucella melitensis biovar suis, helicobacter pylori atcc 43504 and o. intermedium lmg 3301 was done using mega v 6.0 with upgma method (bootstrap = 1000) as described. similarly urease alpha subunit (ure c) of genera ochrobactrum, helicobacter and brucella was used to build upgma tree using mega. genome re - sequencing of m86 strain was done using illumina miseq m02845 sequencer was carried out using 2 300 bp chemistry. to check the quality of paired end poor quality reads (phred quality below q30) and adapter sequences were removed using fastq trimmer and flexbar pre - processing tool respectively. to preserve mate integrity and to filter sequences by composition de novo assembly of prepossessed fastq reads was performed using velvet 1.2.10 (k - mer = 25, 31, 57) and spades 3.0.0 (k - mer 21, 33, 55). further the quality of integrated contigs / scaffolds was evaluated using check bacterial contigs tool. contigs of o. intermedium m86 draft genome and resequenced genome were mapped against chromosome i and ii of o. intermedium lmg 3301 and visualized using brig (fig . to predict gene coding regions ( cds) prokka and rapid annotation subsystem technology (rast) were used. metabolic pathway prediction was performed on kaas to assign kegg orthology (ko) numbers to each predicted cds. comparative analysis was carried out between previous draft genome and resequenced genome for different features such as presence of insertion sequences, tandem repeats, phage genes, clustered regularly interspaced short palindromic repeats (crispr), plasmid and secondary metabolite production genes using is finder, tandem repeats finder, phast, crisprfinder, blast and anti - smash 2.0 respectively. o. intermedium m86 draft genome and resequenced genome were compared with o. intermedium lmg 3301 for single nucleotide polymorphism (snps) detection using parsnp pipeline.

advances in de novo sequencing technologies allow us to track deeper insights into microbial genomes for restructuring events during the course of their evolution inside and outside the host. bacterial species belonging to ochrobactrum genus are being reported as emerging, and opportunistic pathogens in this technology driven era probably due to insertion and deletion of genes. the ochrobactrumintermedium m86 was isolated in 2005 from a case of non - ulcer dyspeptic human stomach followed by its first draft genome sequence in 2009. here we report re - sequencing of o. intermedium m86 laboratory adapted strain in terms of gain and loss of genes. we also attempted for finer scale genome sequence with 10 times more genome coverage than earlier one followed by comparative evaluation on ion pgm and illumina miseq. despite their similarities at genomic level, lab - adapted strain mainly lacked genes encoding for transposase protein, insertion elements family, phage tail - proteins that were not detected in original strain on both chromosomes. interestingly, a 5 kb indel was detected in chromosome 2 that was absent in original strain mapped with phage integrase gene of rhizobium spp. and may be acquired and integrated through horizontal gene transfer indicating the gene loss and gene gain phenomenon in this genus. majority of indel fragments did not match with known genes indicating more bioinformatic dissection of this fragment. additionally we report genes related to antibiotic resistance, heavy metal tolerance in earlier and re - sequenced strain. though snps detected, there did not span urease and flagellar genes. we also conclude that third generation sequencing technologies might be useful for understanding genomic architecture and re - arrangement of genes in the genome due to their ability of larger coverage that can be used to trace evolutionary aspects in microbial system.

epidemiological evidence has supported a protective role for diets low in saturated fat and rich in fruits and vegetables as well as a moderate wine consumption against the development and progression of cardiovascular (cvd) and chronic degenerative disease. the atherosclerotic processes underlying cardiovascular disease are intimately connected with a state of chronic inflammation, involving a variety of pathological changes such as endothelial cell activation, low density lipoprotein (ldl) modification, macrophage chemotaxis, and vessel smooth muscle cell migration. in industrialized societies, this excess in ldl particles promotes transport into the vessel wall where they undergo physicochemical modification, which facilitates their ingress into macrophages ing in foam - cell formation. it is widely recognized that, together with oxidative stress, vascular inflammation, lipid deposition, and smooth muscle cell differentiation, oxidized low density lipoprotein (ox - ldl) level may play a major role in atherosclerosis and cardiovascular disease, and it is a commonly used marker for oxidative damage. in fact, oxidation of the lipids and apoproteins present in ldl leads to a change in the lipoprotein conformation by which ldl is better able to enter the monocyte - macrophage system of the arterial wall and promote the atherosclerotic process. the oxidative modification of ldl convert the native particles ox - ldl into pathogenic, immunogenic, and atherogenic particles. removal of ox - ldl from circulating blood is a promising therapeutic strategy against atherosclerosis and many other diseases. it is widely accepted that the consumption of fruits and vegetables prevents diseases related to the oxidative processes. for instance, individuals at high cardiovascular risk who improved their diet toward a mediterranean diet pattern, reached in antioxidants, showed significant reductions in cellular lipid levels and ldl oxidation. moreover, beneficial effects may partly help to explain the protective cvd effects achieved by foods and beverages containing polyphenols (tea, vegetables, fruits, wine, etc .) , that appears to be mediated via a plethora of biochemical pathways and signaling mechanisms, acting either independently or synergistically (e.g., decreasing ldl oxidation, improving endothelial function, increasing nitric oxide release, modulating inflammation and lipid metabolism, and improving antioxidant status). in particular, the reduction of oxidative stress and inflammation following red wine intake could possibly be due to its polyphenol, and resveratrol, content, as suggested by many studies. red wine (rw) consumption has shown to prevent the acute impairment of endothelial function that occurs following cigarette smoking or consumption of high fat meal and to modulate monocyte migration in healthy subjects. these effects must be considered independent of some of the protective effects of wine linked to ethanol, per se. it has been suggested that the phenolic content of rw may modulate leukocyte adhesion molecules, whereas both ethanol and polyphenols of rw may modulate soluble inflammatory mediators in high - risk patients. the phytoalexin trans-3,5,4-trihydroxystilbene (trans - resveratrol) is present in red grape skins and in wines. synthesis of resveratrol and its derivatives (cis - resveratrol and resveratrol glucoside isomers), called all together stilbenes, is generally formed as natural defense ability of the vines against disease attack. the content of stilbenes in the final product depends mainly on the grape variety, but other factors like canopy management can stimulate the plant's self - defense. focusing on those practices that can modulate both the qualitative parameters in the classical sense (sugars, acidity, etc .), as well as the most truly innovative and related compounds useful to human health, growing methods can get high quality grapes and at the same time stimulate the stilbene synthesis. among these the unpruned vineyards can promote the natural presence of stilbenes too, whose positive role on human health is known. although different studies have reported positive data on gene expression after feeding animals with phenolic rich extracts or normal food, few human studies have shown the potential use of gene expression profiling in blood leukocytes to study the effects of meal consumption and the intake of red wine from nonpruned vineyard (npvrw), on gene expression modulation of oxidative stress, inflammatory, and drug metabolism pathway, in association with ox - ldl. more understanding of the role of npvrw intake on oxidation of lipoproteins may allow rationally targeted dietotherapy that can beneficially alter postprandial biomarkers associated with cvd. to our knowledge, no research has compared the acute effects of npvrw in association with a high fat meal on postprandial biomarkers associated with cvd, including oxidative stress and anti - inflammatory and antioxidant activity. therefore, we set up a randomized, crossover, and controlled dietary intervention trial in healthy human volunteers to evaluate and compare the effects on ldl oxidative status after a mcdonald meal (mcdm), alone or combined with the intake of 30 g alcohol from 250 ml npvrw. moreover, we investigated the gene expression of 208 gene related to oxidative stress, inflammation, and drug metabolism in the same conditions. we hypothesized that the magnitude of changes in postprandial responses would be less with the mcdm associated with npvrw, compared with the mcdm alone. this comparison aims to determine if resveratrol is implicated in the nutrigenomic effects of rw. the first aim of the present study was to assess the ox - ldl level in healthy subjects after the intake of 30 g alcohol from 250 ml npvrw, alone or combined with a mcdm. the second outcome was to examine changes of gene expression levels of 75 genes related to oxidative stress pathways (hosp), 72 of human inflammasome pathways (hip), and 61 genes of human drug metabolism (hdm) in the same conditions. as control, we repeated the same analysis after the intake of 30 g alcohol from 250 ml npvrw fasting, with respect to baseline. after the enrollment (a), the study was conducted with four intervention arms: (b) baseline; (c) mcdm; (d) mcdm + 250 ml npvrw (mcdm + npvrw); (e) 250 ml npvrw. each arm was followed by a three - week washout period to avoid additive effects on treatments to follow. a total of 30 participants were consecutively recruited from those who participated in routine medical check - up at the section of clinical nutrition and nutrigenomic, at the university of rome tor vergata. all subjects were healthy and had no evidence of chronic disease. to be eligible for the study, participants had to meet the following inclusion criteria: age between 18 and 65 years, with a bmi 19 kg / m. exclusion criteria were as follows: pregnancy, active smoking, arterial hypertension (140/90 mm hg), body mass index (bmi) > 30 kg / m, acute or chronic diseases, autoimmune disease hiv / aids, neoplastic disease, intestinal disorders, vegetarianism, and use of antioxidants or vitamin supplements or any medication that could influence inflammation and oxidative stress. subjects daily consuming flavonoid - rich beverages, such as tea, herb tea, coffee, cocoa, and fruit juice, more than 500 ml (as estimated from food frequency questionnaire), were also excluded. subjects were asked to exclude alcoholic beverages 15 days before the first intervention (run - in period) and during the study. natural foods rich in antioxidants intake were monitored so that individual diets had similar antioxidant contents throughout the study. at baseline, the clinical evaluation is based on anthropometric and body composition evaluation, quantification of ox - ldl, and a genomic evaluation of 208 genes belonging to the pathway of oxidative stress, inflammation, and drug metabolism. subjects were asked to maintain their usual lifestyle habits and to report any illness or abnormality presented during the study period. at the end of each arm of the study design, a clinician assessed any adverse effects from the interventions by going through a checklist of symptoms, including bloating, fullness, or indigestion, altered bowel habit, dizziness, and other symptoms that were possibly associated with the interventions. an informed consent was signed by all participants, in accordance with principles of the declaration of helsinki. nutritional status assessment, genomic analysis, settings and data collection were performed at the section of clinical nutrition and nutrigenomic, department of biomedicine and prevention of the university of rome tor vergata. after a 12-hour overnight fast, all subjects underwent anthropometric evaluation. all the individuals were instructed to take off their clothes and shoes before undergoing the measurements. waist and hip measures were taken using a flexible steel metric tape to the nearest 0.5 cm, with subjects standing with arms relaxed by their side and balanced on both feet. hip circumference was also measured according to international society for the advancement of kin anthropometry protocol taken at the greatest posterior protuberance of the buttocks. waist circumference was measured just above the iliac crest as recommended in the national institute of health guidelines. body weight (kg) was measured to the nearest 0.1 kg, using a balance scale (invernizzi, rome, italy). height (m) was measured using a stadiometer to the nearest 0.1 cm (invernizzi, rome, italy). body mass index (bmi) was calculated using the formula: bmi = body weight / height (kg / m). body composition analysis was assessed by dxa (i - dxa, ge medical systems, milwaukee, wi, usa), according to the previously described procedure, for evaluating soft tissues, that is, total body fat (tbfat) and total body lean (tblean). the technique combined a total body scanner, an x - ray source, an internal wheel to calibrate the bone mineral compartment, and an external lucite / aluminium phantom to calibrate the fat compartment. individuals were asked to remove all clothing except for undergarments including shoes, socks, and metal items prior to being positioned on the dxa table. the entire body was scanned beginning from the top of the head and moving in a rectilinear pattern down the body to the feet. the coefficient of variation (coefficient of variation = 100 sd / mean) intra- and intersubjects ranged from 1% to 5%. the coefficient of variation for bone measurements is less than 1%; coefficient of variation on this instrument for five subjects scanned six times over a nine - month period was 2.2% for tbfat and 1.1% for tblean. total body fat percentage (pbf) was calculated as tbfat mass divided by total mass of all tissues, considering also the total body bone (tbbone), as follows:pbf=(tbfat+tblean+tbbone)100. blood samples (10 ml) were collected into sterile tubes containing edta (vacutainer). all materials were immediately placed on ice and plasma was separated by centrifugation at 1600 g for 10 min at 4c. laboratory tests included fasting glucose, hdl - cholesterol, ldl - cholesterol, triglycerides, aspartate aminotransferase (ast), alanine transaminase (alt), creatinine, fibrinogen, and c - reactive protein (crp) levels and were recorded at baseline. all clinical chemistry analyses, except plasma glucose, serum lipid, crp, and triglycerides analysis, were carried out with an advia 1800 chemistry system (siemens healthcare), according to standard procedures. plasma glucose concentrations were measured using the glucose oxidase method with an automated glucose analyzer (cobas integra 400, roche diagnostics, indianapolis, in, usa); serum lipid profile components were determined by standard enzymatic colorimetric techniques (roche143 modular p800, roche diagnostics, indianapolis, in). serum triglycerides were measured on the beckman synchron lx20 automated system by a coupled enzymatic method that produces a red - colored complex. all tests were performed using the same lot of reagents or assay plates in order to minimize variability due to differences in reagent lots. analyses were carried out at the accredited clinical chemical laboratories of the tor vergata hospital of rome, italy. blood sample was collected and stabilized in paxgene blood rna tubes (pre analytix qiagen, hombrechtikon, switzerland) and stored at 80c until rna extraction. the total rna of each collected sample was purified using the paxgene blood mirna kit according to the manufacturer's instructions (preanalytix qiagen, hombrechtikon, switzerland). aliquots of total rna were then quantified and assessed for quality by spectrophotometry (nanodrop, wilmington, usa) and agarose gel electrophoresis. we used specific rt2 profiler pcr arrays (qiagen, netherlands): we focused on the human oxidative stress (pahs-065za, qiagen, netherlands) pathway, human inflammation (pahs-097za, qiagen, netherlands) pathway, and human drug metabolism (pahs-002a, qiagen, netherlands) pathway. each qrt - pcr experiment was performed in triplicate and repeated at least twice, according to manufacturer's instruction (qiagen, netherlands). the value used to plot relative gene expression was determined using the expression fold change (fc) = 2. raw data were filtered for genes that were significantly changed above factor 1.0 within the 95% confidence interval (p 0.05) for each experiment. finally, only genes with an absolute fc value of at least 1.5 and p value 0.05 (indicating a statistical significance) were considered as differentially expressed genes. a fasting blood samples were collected and stabilized in edta and stored at 80c until analysis. circulating ox - ldl level in plasma was measured by enzyme linked immunosorbent assay using the mab-4e6 antibody (mercodia ab, uppsala, sweden), according to the customer protocol. the agricultural trial was started in 2012 on 12-year - old commercial vineyards, located in ponte di piave, treviso, (north - east italy, 454302.93n, 12328:35 e, altitude 3 m asl). the variety was cabernet sauvignon, grafted on 1103 p, and the vineyard has a density of 3300 plants per hectare. the adopted training system was the free cordon (a permanent spur cordon pruning with no wire above the cordon); no pruning (np) treatment have been provided. for the np treatment a randomized block design was applied, with 3 plots, each one consisting of 2 rows (one per treatment). fifteen vines per treatment were chosen and replicated 3 times; the production of grapes was weighed, and the average weight of the bunch was made; on a sample of berries the polyphenol content, total and extractable, according to the methodology proposed by di stefano and cravero, soluble solids content (brix), total acidity (ta), and ph were determined. the contents of some stilbenes, resveratrol, trans and cis, and trans - piceid were determined according to the methodology proposed by sun et al.. the selected dietary treatment was a high fat meal represented by a mcdonald's meal (mcdm) consisting in number 1 big tasty bacon and number 1 small french fries (26.8% of total kcal from carbohydrates ; 18.2% of total kcal from protein, of which about 70% was comprised of animal proteins ; 55% of total kcal from total fat). the bromatological composition of mcdm was obtained using diet analyser software package dietosystem (ds medica srl, milan, italy, version 12.00.13 .). atherogenic index (ai), a parameter to determine the atherogenic risk of a diet, was calculated by the ratio between saturated (c14 : 0, c16 : 0, c18 : 0) and unsaturated fat acids (monounsaturated fatty acid ( mufa) and polyunsaturated fatty acid (pufa)):ia=4c14: 0+c16: 0+c18: 0mufa+pufa6+pufa3.thrombogenicity index (ti) was calculated by the ratio between saturated fat acids (c14 : 0, c16 : 0, c18 : 0) and the sum of mufa and pufa:it = c14: 0+c16: 0+c18: 00.5mufa+0.5pufa6-fatty acids3-fatty acidspufa + 3pufa3 fatty acids+pufa 3-fatty acidspufa6-fatty acids1.cholesterol/saturated fatty acids index (csi) can be used as a fast and accurate way to assess the content of saturated fat and dietary cholesterol. the potential atherogenic risk of food referred to the cholesterol and saturated fat contained in the meal; lower index suggests a low probability of incidence of cardiovascular disease:csi=1.01g of saturated fat+0.05mg of cholesterol. a paired t - test or a nonparametric wilcoxon test was performed to evaluate differences before and after nutritional intervention. in all statistical tests performed, the null hypothesis (no effect) was rejected at the 0.05 level of probability. the differences between ox - ldl levels in the intervention arms were calculated as follows. % = (( z y)/y ) 100, where % is the percentage variation of oxidation of ldl, calculated as ratio of absolute variation to the base value; in particular z is the value of oxidation ldl after nutritional intervention (npvrw, mcdm, and mcdm + npvrw) and y is the value of oxidation if ldl at the nutritional intervention of reference (b and mcdm). the value used to plot relative gene expression was determined using the expression fold change (fc) = 2. data were normalized and we have considered an absolute fold change at least equal to 2.00 (absolute fc 2.00). transcripts were declared differently expressed only when p < 0.05 and fold changes were either < 0.8 (downregulated) or > 1.2 (upregulated), as previously described. the npvrw treatment reported the following quality parameters: the soluble solids (brix) equal to 19.20, a titratable acidity equal to 7.4 g / l, and a ph as 3.38. regarding the content flavonoids and anthocyanins , the wine showed a content of total anthocyanins equivalent to 541 mg / kg of berry, a content of extractable anthocyanins equal to 317 mg / kg of berry. total flavonoids were detected equal to 1346 mg / kg and a content of extractable flavonoids equivalent to 575 mg / kg of berry. the macronutrient content of mcdm was 1144.04 kcal, 81.89 g of carbohydrates, 51.99 g of proteins (40.78 g of animal proteins, 10.60 g of vegetal proteins), 68.89 g of fats (of which 21.71 g saturates, 30.71 g monounsaturates, and 13.96 g unsaturates), and 3.68 g of dietary fiber (of which 1.51 g soluble, and 2.15 g insoluble fiber). the quality indices of mcdm were 1.97 of ai, 1.73 of ti, and 31.05 of csi. of the 30 initial participants initially enrolled, 24 subjects were eligible for the study. four subjects declined to participate during the first phase, and another two did not meet the inclusion criteria: one of them measured a bmi > 25 kg / m, one had a history of heart attack. in tables 1 and 2 the nutritional status characteristics and blood parameters of subjects are shown, before nutritional intervention. the value of obese subjects was obtained by dxa: total percentage of obese was estimated as a 58.33% of total subjects: 71.43% of female and 40.00% of men. according to bmi, 16.67% of total subjects were sarcopenic: 20.00% of male and 14.29% women were sarcopenic. the comparison of ox - ldl level in the nutritional intervention was shown in figure 2. a significant increase (p 0.05) of ox - ldl level was observed between b andmcdm, showing a % = 17.56%. the ox - ldl levels significantly decreased (p 0.05) comparing mcdm versus mcdm + npvrw (% = 20.94%). no significant value (p > 0.05) was highlighted in b versus mcdm + npvrw (% = 7.06%) and in b versus npvrw (% = 5.48%). in particular, we focused on different expression levels of 75 genes of oxidative stress, 72 genes of inflammasome and 61 genes of human drug metabolism pathways (see supplement table s13 in supplementary material available online at http://dx.doi.org/10.1155/2015/317348). the different fold change levels were analyzed for the condition: (i) b versus mcdm; (ii) b versus npvrw; (iii) b versus mcdm + npvrw; (iv) mcdm versus mcdm + npvrw. after data normalization, considering an absolute fold change at least equal to 2.00 (absolute fc 2.00), we identified differential gene expression levels for each condition, as shown in figure 3. in figures 4, 5, and 6 we showed the most representative genes of the studies pathways; a fold change > 2.00 was considered significant (p 0.05). in figure 4 genes of inflammasome pathway are reported, comparing: (i) b versus mcdm; (ii) b versus npvrw; (iii) b versus mcdm + npvrw; (iv) mcdm versus mcdm + npvrw: casp8, caspase 1 apoptosis related cysteine peptidase (nm_1228); ccl2, chemokine (c - c motif) ligand 2 (nm_2982); il6, interleukin 6 (interferon beta 2) (nm_600); nfkb1, nuclear factor of kappa light polypeptides gene enhancer in b - cells 1 (nm_3998). in figure 5 genes of oxidative stress pathway are reported, comparing (i) b versus mcdm; (ii) b versus npvrw; (iii) b versus mcdm + npvrw; (iv) mcdm versus mcdm + npvrw: cat, catalase (nm_1752); ccl5, copper chaperone for superoxide dismutase (nm_2985); gpx1, glutathione peroxidase 1 (nm_581); gpx2, glutathione peroxidase 1 (gastrointestinal) (nm_2083); sirt2, sirtuin 2 (nm_12237); sod1, superoxide dismutase 1 soluble (nm_454); txn, thioredoxin (nm_3329); ucp2, uncoupling protein 2 (mitochondrial proton carrier) (nm_3355). in figure 6 genes of human drug metabolism pathway are reported, comparing (i) b versus mcdm; (ii) b versus npvrw; (iii) b versus mcdm + npvrw; (iv) mcdm versus mcdm + npvrw: adh4, alcohol dehydrogenase 4-class 2 pi polypeptide (nm_670); aldh1a1, aldehyde dehydrogenase 1 family member a1 (nm_689); alox5, arachidonate 5-lipoxygenase (nm_698); nat1, n - acetyltransferase 1 (arylamine n - acetyltransferase) (nm_662); nos3, nitric oxide synthase 3 (endothelial cell) (nm_603). in the supplementary tables the fold change of all analyzed genes and related statistical significance were reported (see tables s13). dietary patterns have been associated with several cardiovascular risk factors such as blood pressure, obesity, serum lipids, and inflammatory markers. in postprandial state, cellular structures such as proteins, carbohydrates, nucleic acids, and lipids are damaged by oxidative processes. high fat diets are contributory atherosclerosis risk factors, since it can cause an acute modification in endothelial function. considering that after each meal, blood concentrations of glucose and lipids are raised, and this postprandial increase lasts for a rather long time; these changes might be of importance in the process of atherosclerosis initiation and progression. moreover, after each meal the production of reactive oxygen species (ros) will lead to the activation of factor nuclear factor-b (nf - kb). this factor is a transcription factor which is contributed in immunity, inflammation, and regulation of cell proliferation, growth, and apoptosis by controlling the expression of many genes. these are all among the mechanisms that might be contributed in the progression of atherosclerosis. although native ldl is exposed to all enzymatic and nonenzymatic oxidants, they are protected by a potent array of antioxidants in plasma. moreover, the ldl and other biomolecules are protected from free radical attack by the action of antioxidant capacity in the blood. the protective effects of enzymatic and nonenzymatic antioxidant activity are exerted in a network manner. therefore, plasma antioxidant status is the of interaction and cooperation of various antioxidants. a decrease in ox - ldl was reported after consumption of beverage as source of polyphenols, such as grape juice, red wine, green tea, and cocoa drink and cranberries. micronutrients modulate immune system and exert a protective action by reducing ldl - cholesterol oxidation via induction of antioxidant enzymes. according setorki et al. using high - dose vinegar with have demonstrated that grape seed proanthocyanidins with meal can lower the postprandial oxidative stress through decreasing oxidants concentration and increasing the level of serum antioxidants. moreover, according to observational studies, due to the synergism of different nutrients consumed in a daily diet, it could be difficult to separate out specific effects. however, it was of our interest investigating how polyphenols from rw intake, combined with mcdm, could be associated with circulating markers of oxidative stress and inflammation, then contributing to increase cardiometabolic risk. in our study, npvrw had a notably higher amount of trans - resveratrol and other stilbenes content compared to commercial wine. this might be an indication that less invasive pruning methods (like the extreme case of no pruning) can lead to a higher amount of resveratrol. according to our previous data, in the present study, after the consumption of mcdm with npvrw compared to mcdm, we observed a significant decrease of ox - ldl levels, with protective effect towards ldl oxidation. the opposite is observed for the mcdm compared to b. moreover, no significant effect on ox - ldl was highlighted after npvrw intake. the ox - ldl level, combined with transcriptomics analysis gives us further insights to understand the global effect of dietary patterns on health. in this study, mcdm was related to biological pathways associated with oxidative stress, inflammatory, and drug metabolism response. opposite to npvrw intake effect, after consumption of mcdm and mcdm + npvrw, we observed an upregulation of nf-b-1 gene expression, probably due to the consumption of a high fat meal. nf-b is the master regulator of the immune / inflammatory response; by regulating nf-b, sirtuin-1 (sirt-1) may inhibit the expression of genes involved in inflammation that is directly related with the nf-b signaling pathway. in addition, an increasing number of studies suggest that resveratrol plays an important role in protecting mitochondria and attenuating mitochondrial ros production, improving endothelium function, inhibiting inflammatory processes and decreasing the rate of endothelial apoptosis. activation of the redox - sensitive nf-b is believed to be an important component of the cascade of events triggered by psychosocial stress, leading to inflammation, thrombosis, and vascular damage. nf-b subunits are expressed ubiquitously and can be activated by a wide range of stimuli, such as ros, cytokines, infection, and dna damage; however, their actions are regulated in a cell- and stimulus - specific manner, leading to a diverse spectrum of effects. sirt 1, activated through both calorie restriction and resveratrol, mediates the beneficial impact of each on longevity and health, acting by deacetylating transcription factors, such as the tumor suppressor p53, the forkhead box (fox) family of transcription factors foxo1, foxo3, and foxo4 and the transcription factors nf-b. it has therefore been suggested that resveratrol mimics the protective effects of dietary restriction, since it can activate the mammalian sirtuin in vitro assays. we observed different levels of sirt2 expression after consumption of the npvrw and mcdm + npvrw, compared to b, probably owed to the resveratrol present in red wine, as suggested by schirmer et al.. we have observed an upregulation of alcohol dehydrogenase (adh4) in comparison between b and mcdm meal. probably, this is due to the presence of ethanol in bread of mcdm sandwich; in fact in the other condition, adh4 is downregulated and aldehyde dehydrogenase (aldh1a1) is upregulated, according to metabolic pathway of ethanol. monocyte chemoattractant protein-1 (mcp-1), also known as chemokine (c - c motif) ligand 2 (ccl2), is a proinflammatory chemokine and recruits and activates monocytes during the inflammatory response. our , from the comparison between b and mcdm, suggest that ccl2 inhibits the transcription of il-6, according to mandrekar et al., who highlighted the role of ccl2 in the regulation of tnf- and il-6 however, the comparison between b versus npvrw, b versus mcdm + npvrw, and mcdm versus mcdm + npvrw shows a downregulation for each gene, ccl2 and il-6, highlighting a control over inflammation genomic condition. as previously demonstrated, we have underlined the relation between catalase (cat) and ccl5. in a proinflammatory situation, due to the consumption of high fat meal, cat downregulated the expression of ccl5. on the other hand, comparing mcdm versus mcdm + npvrw we obtained a downregulation of cat and an upregulation of ccl5. the positive effect of wine is highlighted in comparison between b and npvrw, such as in comparison between mcdm and mcdm + npvrw, in which txn is upregulated. in fact thioredoxin is critical for redox regulation of nf-b and it has general intracellular antioxidant activity and when upregulated or overexpressed protects against oxidative stress. according to poulsen et al. our show an ucp2 upregulation in b versus npvrw, b versus mcdm + npvrw, and mcdm versus mcdm + npvrw comparison. we concluded that npvrw, in association with a high fat meal, could improve the upregulation of ucp2 preventing mitochondrial oxidative stress and hepatic fat accumulation. our show a downregulation of thioredoxin (txn) gene in b compared to mcdm and a downregulation after mcdm + npvrw of the same gene. trx or txn is a 12 kda multifunctional protein with a redox - active site (cys - gly - pro - cys), which is involved in dithiol / disulfide exchange reaction. furthermore, reduced trx prevents apoptosis via an inhibitory binding to apoptosis signal - regulating kinase 1 (ask-1), whereas this binding is lost when trx is oxidized. an increased trx expression could help to increase the reduction of intracellular proteins and other biomolecules as part of the antioxidant defense. our suggest that downregulation of txn gene may be due to hazelnut supplementation but still it is still unclear what the interaction processes are. however, these data reflect gene expression profile and statistical predictions and need to be confirmed by further research. previous study reported that a simple change of 25% of energy load from fat to carbohydrate in a meal significantly improves postprandial proatherogenic factors. observed in this exploratory study support the scientific evidence regarding the deleterious impacts of a high fat meal, such as the mcdm, which represents a usual meal of western dietary pattern. moreover, our also indicate that expression of genes in pathways related to chronic disease are positively influenced by the enrichment of polyphenol during the meal, as demonstrated by the effects of npvrw intake. a limit of the study was due to the small number of participants, although for genomic studies a numerosity of 8 to 25 subjects is acceptable. in addition, it would be appropriate to evaluate the impact on gene expression of other factors associated with a healthy or unhealthy lifestyle, as the physical activity. moreover, another limit of the study was the short - term treatment, due to high level of quality indexes of mcdm, such as ai, csi, and ti that are referred to a higher amount of saturated fat and related to oxidized ldl and pathogenic factor. since the biomarkers used in our study are not commonly examined in the clinical setting, for example, gene expression of drug metabolism, inflammation, and oxidative stress, our can only lead to limited . however, as previously reported, our data highlighted the positive effect of npvrw intake alone or in association with mcdm on ox - ldl, indicating that the antioxidant potential of the nutrients found in red wine may be an essential component to combatting chronic noncommunicable diseases linked to inflammation and oxidative stress. although more studies are recommended to investigate the role of antioxidant capacity on ldl oxidation and gene expression, our study confirms the need of a long term lifestyle intervention based on healthy diet, combined with a moderate intake of high quality red wine, to improve mechanisms of metabolic and cardiovascular diseases, such as oxidative stress and inflammation. in fact, whereas excessive alcohol consumption is among the leading contributors to morbidity and mortality worldwide, moderate alcohol consumption (2 drinks / day for men and 1 drink / day for women) may have some health benefits. in , the idea that is possible to reduce the cardiovascular stress due to postprandial oxidative stress over the course of a meal may seem trivial; however, these daily insults over time may lead to cvd. consuming rw may be a manner to reduce the magnitude of these daily insults, which potentially will reduce cvd risk. the from this study demonstrate that consuming red wine, naturally enriched with resveratrol, in lower postprandial ox - ldl concentration and lower inflammation, according to the vision of social medicine, for the prevention and control of cvd and chronic degenerative diseases. although the present data do not allow the that npvrw intake has an acute protective value for atherosclerosis, it seems reasonable to conclude that these show the favorable acute effects on some risk factors of atherosclerosis, such as ox - ldl, and overexpression of inflammation and oxidative stress related genes. however, more research is needed on a larger population and on chronic effects of npvrw intake before definitive can be made.

postprandial oxidative stress is characterized by an increased susceptibility of the organism towards oxidative damage after consumption of a meal rich in lipids and/or carbohydrates. micronutrients modulate immune system and exert a protective action by reducing low density lipoproteins (ldl) oxidation via induction of antioxidant enzymes. we evaluated the gene expression of oxidative stress (hosp), inflammasome (hip), and human drug metabolism pathways (hdm) and ox - ldl level at baseline and after the intake of red wine naturally enriched with resveratrol (npvrw), in association with or without a mcdonald's meal (mcdm). the ox - ldl levels significantly increase comparing baseline (b) versus mcdm and decreased comparing mcdm versus mcdm + npvrw (p 0.05). percentages of significant genes expressed after each nutritional intervention were the following: b versus mcdm, 2.88% hosp, 2.40% of hip, and 3.37% of hdmp; b versus mcdm + npvrw, 1.44% of hosp, 4.81% of hip, and 0.96% of hdmp; mcdm versus mcdm + npvrw, 2.40% of hosp, 2.40% of hip, and 5.77% of hdmp; b versus npvrw, 4.80% hosp, 3.85% hip, and 3.85% hdmp. npvrw intake reduced postprandial ox - ldl and the expression of inflammation and oxidative stress related genes. chronic studies on larger population are necessary before definitive .

epidemiological studies reveal that approximately 2.3 billion adults will be overweight and 700 million will be obese in 2015. obesity is a chronic subclinical inflammatory disease of multifactorial etiology, involving the ingestion of a high - calorie diet, a sedentary lifestyle, and genetic predisposition. the establishment of the disease can lead to the development of correlated morbidities such as diabetes mellitus type 2, cardiovascular disease, and metabolic syndrome, among others, as a consequence of the imbalance of a complex system of energy balance. the enlargement of adipocytes and the expansion of adipose tissue lead to local hypoxia and macrophage infiltration, causing an inflammatory response. in turn , the response changes the patterns of protein and gene expression of various bioactive molecules, called adipokines, produced by adipose tissue. adipokines are a group of proteins synthesized by white adipose tissue that act on the immune, cardiovascular, metabolic, and endocrine systems. examples include tnf-, leptin, adiponectin, resistin, il-6, and il-10. in obese individuals, the expression of proinflammatory adipokines by adipose tissues tnf- and il-6 are involved in the development of insulin resistance and atherogenic processes. however, il-10, a cytokine secreted primarily by monocytes / macrophages and lymphocytes, as well as adiponectin, secreted by adipocytes, possess anti - inflammatory and insulin - sensitizing properties by antagonizing il-6 and tnf-. low concentrations of il-10 and adiponectin have been associated with metabolic syndrome and diabetes mellitus type 2. the il-10/tnf- ratio has been considered an important indicator of inflammatory status, and low values are associated with an increased risk of morbidity and mortality. the lipid composition of the diet has a strong relationship with the development and persistence of obesity. acute (2 days) or chronic (16 weeks) treatment with a high - fat diet reduces the synthesis of adiponectin, suggesting that the serum lipid profile and lipid components of the diet are more related to the decrease of adiponectin than to obesity itself. in adult males, a positive correlation between body fat and the quantity or quality of lipids consumed has been described, and diets high in saturated and monounsaturated fatty acids induced greater adiposity compared with diets rich in polyunsaturated fatty acids. studies by our group have shown a link between a high - fat diet and different types of lipids in the diet with changes in gene expression, the synthesis and secretion of adipokines and obesity - related comorbidities. thus, the aim of this study was to investigate the effects of high - fat diets enriched with lard, soybean oil, or hydrogenated vegetable oil (30% lipids, w / w) on body fat content, lipid profiles, serum glucose, insulin and adiponectin concentrations, and the tissue cytokines tnf-, il-10, and il-6. the experimental research committee of the so paulo federal university approved all procedures for the care of the animals used in this study (protocol cep n1644/10). thirty - day - old male swiss mice were used in this study and were kept under controlled conditions (12 h light : 12 h dark cycle at 22c 1c). during the experimental period, the animals were housed six per cage, receiving water and a specific diet ad libitum. the animals were assigned to four experimental groups, with 12 animals per group, according to their specific diet and treated for eight weeks as follows: c (control group ; the animals were treated for eight weeks with a standard ain diet ( american institute of nutrition); s (soybean oil group); l (lard group); and h (hydrogenated vegetable oil group). the animals' weights were evaluated weekly. all diets were prepared according to the recommendations of the ain and were normoproteic. all groups received a growth diet (ain-93 g) containing 20% protein and at least 7% essential fatty acids between the 30th and 60th day of life and a maintenance diet (ain-93 m) containing 14% protein and 2% soybean oil to provide the essential fatty acids between the 60th and 90th days of life. the compositions of the control, soybean, lard, and hydrogenated vegetable oil diets are described in table 1. 500 mg of the prepared diets were used for lipid extraction, saponification, and fatty acid methylation as described by the american oil chemists - society, 2011. the relative fatty acids content in the total lipids from the diet was quantified by gas - liquid chromatography using agilent technologies 7890a cg system, with an ionizable flame detector acoplado linked to the ezchrom elite cds software (agilent technologies, inc . fatty acids were separated using an sp 2560 capillary column ( supelco, usa), with 100 m 0.25 mm 0.20 m measurements. the methylated fatty acids were identified by comparing their retention times to known standards (nu - chek prep . were expressed weight percentage ( g/100 g of total fatty acids table 2). the animals were sacrificed by decapitation on the 90th day of life in the fasting state (8 h) in the early morning to avoid chronobiological variations. trunk blood was collected and immediately centrifuged at 4c, and serum aliquots were taken and frozen at 80c to measure the concentrations of glucose, triacylglycerols, and total cholesterol using commercials kits from labtest diagnostic sa (minas gerais, brazil) and the concentrations of free fatty acids, insulin, and adiponectin using elisa (linco research, inc . , usa). the retroperitoneal (ret), epididymal (epi), and mesenteric (mes) adipose tissues, gastrocnemius muscle (gast), and liver were dissected and weighed, frozen in liquid nitrogen, and stored at 80c until the extraction of protein. was measured as described by stansbie et al. and standardized using the method described by oller do nascimento and williamson. briefly, an eviscerated carcass was autoclaved at 120c for 90 min and then homogenized with double its mass of water. triplicate aliquots of this homogenate were weighed and digested in 3 ml of 30% koh and 3 ml of ethanol for at least 2 h at 70c in capped tubes. after cooling, 2 ml of 12 n h2so4 was added, and the sample was washed three times with petroleum ether for lipid extraction. the are expressed as grams of lipid/100 g of carcass. adipose tissue depots, gastrocnemius muscles, and livers (0.250.3 g) were homogenized in ice - cold solubilization and total protein extraction buffer (100 mm tris, ph 7.5, 10 mm ethylene acetic acid, 100 mm sodium fluoride, 10 mm sodium orthovanadate, 2 mm phenylmethylsulfonyl fluoride, 10 mm sodium pyrophosphate, and 0.1 mg / ml aprotinin). homogenates were centrifuged at 14000 g for 40 min at 4c, the supernatants were saved, and the protein concentrations were determined using a bradford assay (bio - rad, hercules, california) with bovine serum albumin as a reference. the are expressed as the means sem. for multiple comparisons of means, one - way analysis of variance (anova) was performed with subsequent use of the tukey post hoc test. simple linear regression analysis was used to evaluate the correlation between cytokines (il-10 and tnf-). body mass, white adipose tissue depots (epi, ret, and mes), liver, and gast weights were similar among all groups. however, the s and l diets promoted a significant increase in carcass relative lipid content compared with group c. in addition, this parameter in group h was lower than in group s (table 3). exposure to the l diet after weaning caused an approximately 28% increase in the sum of the adipose tissue depots compared with the control group. the levels of serum glucose, triacylglycerols, total cholesterol, insulin, and adiponectin did not differ among the studied groups. nevertheless, the l diet caused a decrease in the serum concentrations of free fatty acids (table 4). tnf- levels did not change in any group or tissue studied compared with the control group (figure 1(a) ). however, the l diet caused a decrease in il-10 levels in mes and ret white adipose tissue depots, and the h diet promoted a similar effect in the ret depot (figure 1(b) ). furthermore, the il-6 content was elevated in mice that ingested the diet containing saturated fatty acids (l) (figure 1(c) ). in group h, the il-10/tnf- ratio was higher in mes adipose tissue compared with groups s and l (figure 1(d) ). we ran correlation tests for il-10 and tnf- and found the following to be positively correlated: group c, retroperitoneal fat depot, liver, and gastrocnemius muscle; group s, epididymal and mesenteric fat depots and gastrocnemius muscle; and group l, liver and gastrocnemius muscle. in group h, only mesenteric fat tissue did not correlate. the ingestion of high - fat diets for 8 weeks did not modify body or tissue weight. similar were observed by others with eight or seven weeks of high - fat diet treatment. however, other studies have reported an increase in body weight after 16 weeks of high - fat diet treatment. as stated by noeman et al., the high - fat diet effect is subtle but cumulative, needing at least 10 weeks to increase body weight. these suggest that the difference in weight gain in the previous studies may be due to the period of treatment. despite the fact that the carcass lipid content in groups s and l was higher than in group c, this parameter was similar between groups c and h. the oxygen consumption after a meal was significantly lower in rats fed a lard diet than in rats fed a safflower oil or linseed oil diet. in this sense, dube et al. demonstrated that the ingestion of a hyperlipidic diet, enriched with lard, caused a decrease in the mrna expression of ucp-1 in brown adipose tissue. furthermore, the energy cost of lipid deposition from dietary fat is lower than from dietary carbohydrate.. demonstrated that enrichment of the diet with polyunsaturated fatty acids caused changes in adipose tissue metabolism, such as the increased uptake of diet - derived lipids favoring fat deposition. machado et al. reported that mice fed with a trans - fat - enriched diet for 16 weeks showed a reduction in subcutaneous and epididymal fat pads, suggesting an effect on the stimulation of lipolysis. taken together, these reports suggest that the effects on fat accumulation depend on the type and source of lipids present in the diet and could be related to the effects on energy expenditure and adipose tissue metabolism, such as lipoprotein lipase activity, lipogenesis, and lipolytic enzyme activities. the diet intervention did not alter the concentrations of serum glucose, triacylglycerol, total cholesterol, adiponectin, or insulin. previous studies have demonstrated no effect of high - fat diets on the serum adiponectin concentration. however, a time - dependent effect of a high - fat diet on the adiponectin serum concentration has been shown; 10 weeks of treatment increased and 18 weeks of treatment reduced the serum adiponectin concentration compared with the initial experimental period, suggesting a compensatory mechanism to maintain metabolic homeostasis, as this adipokine is associated with the maintenance of carbohydrate and lipid metabolism and also acts to improve insulin sensitivity. in fact, in our study, 8 weeks of a high - fat diet treatment did not modify serum glucose, triacylglycerol, total cholesterol, or adiponectin and insulin concentrations, in spite of the increasing adiposity in groups l and s. another possible explanation related to the similar serum lipid profile among the groups is the decrease in the carbohydrate content of the high - fat diets. the review from volek et al. reported that the serum total cholesterol level remained unchanged from baseline values, while both the hdl and ldl cholesterol levels increased and the tag levels dramatically decreased under a high - fat diet compared with high - carbohydrate diets. in the current study, the lard diet caused a decrease in the serum free fatty acid concentration in relation to the control diet. it has been stated that the plasma free fatty acids ed from hydrolyzed triacylglycerol in chylomicrons and vldl by lipoprotein lipase activity and lipolysis in adipose tissue. one important mechanism for the removal of unesterified plasma fatty acids is through oxidation of these fatty acids in skeletal muscle and the heart. several studies have demonstrated that il-6 can increase fatty acid oxidation in myocytes and in vivo. when we analyzed the il-6 content in gastrocnemius muscle, we verified an increase in group l compared with group c, which could partially explain the reduction in serum fatty acids in lard - treated animals. nutrients such as the fatty acids in special saturated fatty acids can activate intracellular pathways through tlr2 and tlr4, leading to increased proinflammatory gene transcription. in this sense, we analyzed the tnf-, il-6, and il-10 concentrations in ret, epi, mes, gastrocnemius muscle, and liver. the literature is controversial about the effects of a high - fat diet on tnf- expression. a similar was observed by flanagan et al., who showed that tnf- gene expression in muscle was not significantly affected by the amount or type of dietary fat. hong et al. showed that 10-week - old mice treated with a high - fat diet (55% fat by calories) for 3 weeks had lower il-10 levels in muscle than control mice, accompanied by a decrease in muscle insulin sensitivity. furthermore, il-10 treatment prevents muscle insulin resistance by decreasing obesity - associated macrophages and cytokines in muscle from high - fat diet - treated mice. in the present study, the il-10 gastrocnemius muscle content did not change in treated mice between 30 and 90 days of life with high - fat diets. however, the lard diet caused a decrease in il-10 in mes and ret, and the same was observed in ret from group h without any change in glycemia or insulin plasma concentration, suggesting that the type of diet, age of the animal, and time of treatment could have different effects depending on the tissues evaluated. in addition, we can not dismiss the possibility that metabolic processes adapt to changes in dietary components during a specific period of life. the il-6 level in gastrocnemius muscle increased in group l compared with group c. the role of il-6 action in obesity - associated insulin resistance remains highly controversial; wunderlich et al. showed that mice with a hepatic deficiency of il-6 receptor a (il-6ra) develop insulin resistance not only in the liver but also in skeletal muscle and wat. in addition, the il-6 level increases with exercise, a situation in which glucose uptake by muscles is high. it is interesting to note that, in the present study, the lard diet, which promoted a decrease in il-10 in adipose tissue depots, caused an increase in il-6 in gastrocnemius muscle. from these , it is possible to suggest that the whole body of an adult animal tries to maintain carbohydrate and lipid metabolism homeostasis by modifying the expression and secretion of cytokines. in fact, by analyzing the ratio between il-10 and tnf-, which is used as an indicator of the inflammatory condition of the individual (where lower values are associated with a poor prognosis) , we verified a similar among groups in all tissues studied. in addition, a positive correlation between tnf- and il-10 was observed in the liver (groups c, l and h) and muscle (all studied groups) (figure 2), which could contribute to the maintenance of glucose, insulin, and lipid profiles at normal values. in summary, our demonstrate that enrichment of the diet with soybean oil or lard elevated carcass lipid content, whereas enrichment with hydrogenated vegetable oil did not alter this parameter as compared to control diet (4% of soybean oil). only the lard diet modified the fatty acid serum concentration, decreased il-10 in adipose tissue depots and increased il-6 in gastrocnemius muscle. these suggested that the type of fatty acid present in the diet plays a part in determining the amount of carcass lipid content and serum fatty acid concentration and influences the cytokines content in the tissues.

this study analyzed the effect of diet enriched with 30% lipids on cytokines content in different tissues. swiss male mice were distributed into four groups treated for 8 weeks with control (c, normolipidic diet); soybean oil (s); lard (l); and hydrogenated vegetable fat (h). we observed an increase in carcass fat in groups s and l, and the total amount of fatty deposits was only higher in group l compared with c group. the serum levels of free fatty acids were lower in the l group, and insulin, adiponectin, lipid profile, and glucose levels were similar among the groups. il-10 was lower in group l in mesenteric and retroperitoneal adipose tissues. h reduced il-10 only in retroperitoneal adipose tissue. there was an increase in il-6 in the gastrocnemius muscle of the l group, and a positive correlation between tnf- and il-10 was observed in the livers of groups c, l, and h and in the muscles of all groups studied. the suggested relationships between the quantity and quality of lipids ingested with adiposity, the concentration of free fatty acids, and cytokine production in white adipose tissue, gastrocnemius muscle, and liver.

an important task in the twenty - first century is to further develop fuel cells as alternative electrochemical devices for efficient generation of electricity. the low - temperature acid - type systems, such as hydrogen - oxygen polymer electrolyte membrane fuel cells and direct alcohol fuel cells, are, at present, the most commonly studied devices in many laboratories worldwide. among organic compound fuels for anodic reactions in fuel cells, methanol has been historically most extensively studied, and more recently, other short chain liquid fuels such as ethanol and ethylene glycol have become important. in the case of direct methanol fuel cells (dmfc), slow electrode kinetics of methanol oxidation and methanol adsorption products (which is mainly coads) poisoning the surface of pt electrode at low temperature still hamper application. as a , polyhydric and monohydric (except methanol) alcohols, such as ethylene glycol and ethanol, have been proposed as potential fc fuels which are much less volatile and less toxic than methanol. moreover, both alcohols have some of the largest volumetric energy densities, and they involve the transfer of a number of electrons that set a practical challenge for the effectiveness of catalysts. they also show lower permeability through membranes (lower crossover effect). however, the electrochemical oxidation of short chain alcohol (monohydric or polyhydric alcohols) is much more complex than, for example, h2 oxidation. the main challenge generally for electrooxidation of polyhydric and monohydric alcohols to carbon dioxide is associated with the cleavage of the c c bond for complete conversion. due to incomplete oxidation, various intermediate species from electrooxidation in both alcohols it is well established that pt is rated as the most active material for oxidation of small organic molecules in acidic media, but poisoning by the intermediate by - product of co - adsorbed species of the binary or ternary platinum - based alloys with ru, sn, rh, pb, w, or mo was proposed to enhance the electrooxidation activity toward alcohols. alternatively, ir has also been employed as a co - metal for platinum - based catalysts in unitized polymer electrolyte fuel cells because of its high stability and resistance to corrosion. the presence of ir, particularly iro2, enhances the electrooxidation of methanol in direct methanol fuel cells due to providing a large number of oh groups that are adsorbed at relatively low potentials. moreover, cao et al. reported that the addition of ir into sn showed to be a promising alternative for pt - based catalysts for ethanol electrooxidation. demonstrated a positive effect for ethanol oxidation in which iro2 was incorporated to platinum - based catalysts. recently, adzic et al. revealed that the presence of a high content of ir atoms into ternary catalyst ptir / sno2/c enhances the complete electrooxidation of ethanol to co2 at a relatively low - onset potential. furthermore, ptir catalysts have been utilized for electrooxidation of ethylene glycol with positive . one of the numerous approaches to increase the electrocatalytic activity of platinum - based catalysts toward the oxidation of small organic compounds is the use of transition metal oxides as support systems for catalytic metal sites. the presence of transition metal oxides in the neighborhood of catalytic sites of noble metal catalysts in an increasing population of oh groups at low potentials, thereby mitigating co poisoning of catalytically active platinum centers, possibly facilitating the cleavage of c this assumption is in accord with reports in which a significant improvement in oxidation of small organic molecules with metal oxides (e.g., wo3, moo3, tio2, zro2, v2o5, and ceo2) modified by pt - based alloy catalysts has been observed. the present work will concentrate on the preliminary investigation of a carbon - supported ptir - based anodic catalyst with tungsten oxide as the additive prepared by the adsorption of tungsten acid. not only the peak current value during cyclic voltammetry (cv) tests in a broad potential region, but also the more specific performance in the low potential region will be evaluated from a more comprehensive point of view. thus, the electrocatalytic activity toward ethanol and ethylene glycol oxidation in comparison with that of ptir / c and wo3-modified ptir / c catalysts will be evaluated by cv, linear sweep voltammetry (lsv), and chronoamperometry (ca) methods. all chemicals were commercial materials of analytical grade purity that were obtained from premetek ptir / c nanoparticles (20 % on vulcan xc-72, pt : ir 1:1). solutions were prepared using doubly distilled and subsequently deionized (millipore milli - q) water. argon was used to de - aerate the solutions and to keep an oxygen - free atmosphere over the solution during the measurements. some characteristics of catalytic particles were obtained using a libra 120 transmission electron microscope (tem) operating at 120 kv. samples for tem measurements were prepared by depositing drops of colloidal solutions of nanoparticles onto 400-mesh copper grids supporting a fromvar film (agar scientific) and, later, drying them in ambient laboratory conditions (temperature, 20 2 c) for 24 h prior to tem analysis. x - ray diffraction (xrd) patterns of the catalysts were obtained with a bruker d8 discover operating with a cu x - ray tube and vantec (linear) detector (k = 1.5406). the working electrode was glassy carbon, and the counter electrode was carbon rod. as a rule, all potentials in the present work were measured versus a k2so4-saturated hg2so4 reference electrode and were recalculated and reported versus the reversible hydrogen electrode (rhe). the catalyst layer was fabricated through modification of the working electrode by immobilization of ptir / c nanoparticles. wo3 modification of the ptir / c catalyst was in accordance with the procedure described in our previous papers. briefly, a solution of tungstic acid was prepared by passing an aqueous solution of 0.05 mol dm na2wo4 through a proton exchange resin. in a typical procedure, selected amount of ptir / c catalyst was added to 2 cm of 0.05 mol dm aqueous solution of tungstic acid. the ing suspension was stirred for 24 h. during that process, the ptir / c nanoparticles interacted with tungstic acid to form tungsten oxide or hydrogen tungsten oxide bronzes. the supernatant solution was centrifuged and replaced with water in order to obtain a colloidal solution of tungsten oxide - modified ptir / c nanoparticles that was stable for months. the pt - to - tungsten ratio in the given catalyst system was determined with x - ray fluorescence (xrf); the targeted ratio was approximately 1:1. to prepare a homogeneous catalyst layer on the glassy carbon working electrode surface, a 5-l aliquot of the catalyst dispersion was deposited using a micropipette (the nominal loading of catalyst was approximately 160 g cm) and allowed to dry under ambient conditions. prior to this step, when the catalyst layers had dried, 2 l of nafion (0.02 % alcoholic solution) was dropped on top of the glassy carbon electrode surface covered with the catalyst and dried at room temperature. prior to the electrooxidation processes, the catalytic electrodes were scanned with 25 complete oxidation / reduction cycles between 0.0 and 0.8 v in 0.5 mol dm h2so4 at 50 mv s scan rate. the co - stripping experiments were carried out in 0.5 mol dm h2so4 electrolyte utilizing the glassy carbon electrode substrate onto which surface the appropriate catalyst was introduced. as a rule, a few cyclic voltammetric measurements (at 50 mv s) were recorded in the potential range from 0.0 to 0.8 v in the deoxygenated electrolyte. the co - saturated solution was prepared by flowing pure co (from air liquide) through the electrolyte for 10 min. the co adsorption process that was employed (mainly on the surface of catalytic pt nanocenters) was achieved by underpotential control at 0.1 v versus rhe for 5 min, after which the dissolved co was removed from the electrolyte by bubbling argon for 30 min maintaining the applied potential (0.1 v), in order to have a solution free of co. then, the adsorbed co monolayer was stripped by recording three cyclic voltammetric scans in the potential range from 0.0 to 0.9 v at a scan rate of 10 mv s. the measurements using these catalysts were repeated three or four times with freshly prepared electrodes, and the average are presented here. the x - ray patterns of the ptir / vulcan nanoparticles in the presence and absence of the wo3 modifier are shown in fig. 1a, b. the broad diffraction peak centered at 20.025.0 in all the xrd pattern is attributed to the hexagonal carbon support. in the case of unmodified ptir 39.5, 46.3, and 67.7 correspond to the pt lattice planes (pcpdf 04 - 0802). the diffractogram of the wo3-modified ptir / vulcan electrocatalyst shows peaks at 2 = 40.5, 46.9, and 66.2, which are associated with pt reflections and the signals that originated from wo3 (pcpdf 43 - 0679). the indicate that pt fcc is the main crystalline phase in the catalysts and that the presence of tungsten species ed in the formation of crystalline aggregates. a peak shift is observed which could indicate the interaction between ptir / vulcan alloy and tungsten oxide. of the xrd patterns, none were metallic ir, or iridium oxide diffraction peaks have been observed because pt and ir metals have similar diffraction peak positions and crystalline structures. to estimate the average particle sizes from scherrer s equation, the pt peak (at 2 = 39.5) was used. the latter peak was chosen because it is located in a region where there are no interferences from the carbon support. the average pt particle sizes were obtained from the position and the full - width at half - maximum values of the pt peak (at 2 = 39.5). the values were in the ranges from 4 to 6 and 6 to 8 nm for unmodified and modified ptir / vulcan nanoparticles, respectively. higher values of the average particle sizes for the wo3-modified ptir / vulcan can be interpreted in terms of deposition of the wo3 crystalline monoclinic structure.fig. 1xrd diffractograms of ptir / vulcan (a) and wo3-modified ptir / vulcan (b) xrd diffractograms of ptir / vulcan (a) and wo3-modified ptir / vulcan (b) in order to get more information about the size, morphology, and distribution of nanoparticles on the carbon material, tem analysis was performed. figure 2 shows the tem images and distributions of the series of ptir / c and wo3-modified ptir / c nanoparticles. low magnification images show that the supported material was predominantly irregular spheres or spheroids of bimetallic nanoparticles that were homogeneously dispersed on the carbon (vulcan xc-72) surfaces. the average particle sizes lie in the narrow range of 58 nm with a standard deviation of 1 nm, which is in agreement with the xrd for wo3-modified ptir / vulcan and unmodified ptir / vulcan catalysts.fig. 2low magnification micrographs (tem) of ptir / vulcan (a) and wo3-modified ptir / vulcan (b) low magnification micrographs (tem) of ptir / vulcan (a) and wo3-modified ptir / vulcan (b) for initial electrochemical characterization, cyclic voltammetric curves of the ptir / vulcan and the wo3-modified ptir / vulcan nanoparticles deposited on glassy carbon electrode were obtained in 0.5 mol dm sulfuric acid - supporting electrolyte (fig . 3). in both cases, slight changes in the shape or current values of the cyclic voltammetric curves are observed. in the hydrogen adsorption / desorption region (between 0.0 and 0.4 v versus rhe) for the all proposed catalysts, some changes in the voltammetry are seen because of the dependence on the surface composition.. 3a ) is characterized by a single large peak in the hydrogen adsorption / desorption region, whereas the electrochemical behavior of electrodes made from the wo3-modified ptir / vulcan nanoparticles (fig . . moreover, in the double layer region, which is between 0.4 and 0.7 v versus rhe, significant currents are recorded . this behavior is typical of electrocatalysts composed of transition metals dispersed on a carbon black support . it is apparent from fig . 3b that the oxidation peak appearing at about 0.2 v most likely reflects the intercalation of protons in tungsten oxide ( wo3) and tends to overlap with the hydrogen adsorption / desorption region of bare platinum (at a potential lower than 0.4 v). this interpretation is difficult to prove because of the problem of unambiguously distinguishing contributions from the reversible reduction of tungsten oxide to hydrogen tungsten bronzes from the abovementioned hydrogen adsorption and desorption peaks originating from pt and ir.fig. 3cyclic voltammetric responses of ptir / vulcan (a) and wo3-modified ptir / vulcan (b) catalytic systems in 0.5 mol dm h2so4. scan rate = 10 mv s cyclic voltammetric responses of ptir / vulcan (a) and wo3-modified ptir / vulcan (b) catalytic systems in 0.5 mol dm h2so4. scan rate = 10 mv s the behavior of the proposed catalytic layers was tested toward ethanol and ethylene glycol electrooxidation processes. representative cyclic voltammograms of these species at both ptir / vulcan and wo3-modified ptir / vulcan electrocatalysts deposited on glassy carbon electrode are shown in fig. 4b ) obtained at the ptir / vulcan and wo3-modified ptir / vulcan surface, respectively, show well - defined peaks for both forward and reverse scans in the investigated potential region. in the forward scan, the ethanol oxidation current at ptir / vulcan catalysts starts (at 0.3 v) and reaches the peak at 0.87 v, which is located at the same potential as compared to the wo3-modified ptir / vulcan, as illustrated in fig. , a single peak is developed at 0.7 v, which can be attributed to the oxidative decomposition of by - products.fig. 4cyclic voltammetric responses for oxidation of 0.5 mol dm ethanol (a) with ptir / vulcan (a) and wo3-modified ptir / vulcan (b) and 0.5 mol dm ethylene glycol (b) with ptir / vulcan (a) and wo3-modified ptir / vulcan (b) catalysts. scan rate = 10 mv s cyclic voltammetric responses for oxidation of 0.5 mol dm ethanol (a) with ptir / vulcan (a) and wo3-modified ptir / vulcan (b) and 0.5 mol dm ethylene glycol (b) with ptir / vulcan (a) and wo3-modified ptir / vulcan (b) catalysts. scan rate = 10 mv s similar measurements to those presented in fig. the cyclic voltammograms of wo3-modified ptir / vulcan and bare ptir / vulcan in the presence of ethylene glycol exhibited no significant oxidation current up to 0.27 and 0.35 v, respectively. two distinct peaks were observed in the potential range between 0.4 and 0.9 v in comparison to previous literature reports , and these processes can be attributed primarily to the oxidation of adsorbed organic species. in the case of electrooxidation of ethylene glycol, various intermediate oxidation products can be expected according to spectroscopic data including co, glycol aldehyde, glycolate, glyoxylate, oxalate, and formate. this is in contrast to the data obtained with ethanol, the oxidation of which produces only ch2cooh, co2, and ch3cho. generally, in the presence of both fuels, the current density in the hydrogen region decreases in comparison to the voltammograms obtained in the supporting electrolyte alone due to their adsorption. additionally, fig. 4 displays that in both cases, the current density values are higher in the tested potential range confirming the enhancement of the electrooxidation of ethanol and ethylene glycol by the presence of the metal oxide wo3. in addition, the onset potentials of ethanol and ethylene glycol oxidation shift toward more negative values which is especially pronounced in the case of ethylene glycol oxidation. this can be explained by the fact that transition metal oxides (e.g., wo3 and related compounds) are known to activate interfacial water molecules (from oh groups on wo3) at lower potentials which, in turn, promote the removal of poisoning species from the noble metal catalyst. figure 5 exhibits - subtracted linear scan voltammograms (lsvs) for ethanol and ethylene glycol electrooxidation on ptir / vulcan and wo3-modified ptir / vulcan electrocatalysts deposited on glassy carbon substrate recorded in the potential range of 0.00.9 v. as can be seen for both fuels, the shape of the lsv curves is almost the same in the examined potential range. the only difference is that the current densities are higher for wo3-modified ptir / vulcan nanoparticles than those recorded on bare ptir / vulcan nanoparticles. the lsv experiments also confirm the shifting of the onset potential for ethanol and ethylene glycol electrooxidation on wo3-modified ptir / vulcan catalysts toward lower potential values. it is likely that the increases of the oxidation current densities observed in the lsv curves are associated with the addition of tungsten oxide. the decrease of the onset potential of ethylene glycol and ethanol oxidation is due to activation of interfacial water molecules forming oh species at lower anodic potential than the bare catalyst.fig. 5linear scan voltammetry responses for oxidation of 0.5 mol dm ethanol (a) with ptir / vulcan (a) and wo3-modified ptir / vulcan (b) and 0.5 mol dm ethylene glycol (b) with ptir / vulcan (a) and wo3-modified ptir / vulcan (b) catalyst at 10 mv s scan rate. electrolyte = 0.5 mol dm h2so4 linear scan voltammetry responses for oxidation of 0.5 mol dm ethanol (a) with ptir / vulcan (a) and wo3-modified ptir / vulcan (b) and 0.5 mol dm ethylene glycol (b) with ptir / vulcan (a) and wo3-modified ptir / vulcan (b) catalyst at 10 mv s scan rate. electrolyte = 0.5 mol dm h2so4 the effect of temperature for ethanol and ethylene glycol electrooxidation in bare and wo3-modified ptir / vulcan nanoparticles was also investigated. when the temperatures increase from 10 to 50 c, the oxidation current densities for both become higher. based on the arrhenius equation, the activation energy (45 and 49 kj mol, respectively) can be determined from the slope calculated by linear regression by plotting ln(j) as a function of reciprocal of temperature (figure not shown). the value of activation energy is in good agreement with a previous report on alcohol electrooxidation. this observation suggests that the increase of temperature causes more facile oxidation kinetics and decreases poisoning by the intermediate species. in order to evaluate the electrocatalytic activity and also the long - term stability of tungsten oxide - modified ptir / vulcan nanoparticles for ethanol and ethylene glycol oxidation, chronoamperometric measurements were performed at low potentials (fig . the polarization current density for the electrooxidation of both fuels on the investigated catalytic systems displays a rapid decrease in the first period of the experiment before reaching a stable value . the catalytic current developed for tungsten oxide - modified ptir / vulcan electrocatalysts for both ethanol and ethylene glycol always was significantly higher than those produced for bare ptir / vulcan nanoparticles in the tested time period . these are consistent with those obtained by cyclic voltammetry . in the initial phase of the chronoamperometric experiments, it is likely that a higher number of free active sites are available for adsorbed ethanol or ethylene glycol molecules ( fast kinetic rate reaction), and during the next few minutes (rate determining step), the amount of free catalyst sites is limited by poisoning by intermediate species, such as co, chx, ch3cho, ch3cooh (for ethanol oxidation), glycol aldehyde, glycolate, glyoxylate, oxalate, and glycolate (for ethylene glycol). in this regard , the improvement of catalytic properties observed by introduction of wo3 on pt - based nanoparticles surface can be associated with the oxophilic nature of tungsten oxide providing hydroxyl groups (oh) on the oxide surface at lower potential, which promotes electrooxidation of the surface co - poisoning intermediates species.fig. 6chronoamperometric current - time responses (recorded at 0.3 v) for oxidation of 0.5 mol dm ethanol (a) with bare ptir / vulcan (a) and wo3-modified ptir / vulcan (b) and 0.5 mol dm ethylene glycol (b) with ptir / vulcan (a) and wo3-modified ptir / vulcan (b) catalysts. electrolyte = 0.5 mol dm h2so4 chronoamperometric current - time responses (recorded at 0.3 v) for oxidation of 0.5 mol dm ethanol (a) with bare ptir / vulcan (a) and wo3-modified ptir / vulcan (b) and 0.5 mol dm ethylene glycol (b) with ptir / vulcan (a) and wo3-modified ptir / vulcan (b) catalysts. electrolyte = 0.5 mol dm h2so4 regarding the stability of the electrocatalytic responses in the presence of ethylene glycol and ethanol, the long - term chronoamperometric and repetitive voltammetric measurements of wo3-modified ptir / c and bare ptir / c systems have been performed (not shown here) under the same conditions. in the potential range between 0.0 and 0.9 v, the catalytic peak currents decreased, remaining at 90 % than those of the first cycle after 100 cycles when the wo3-modified system has been used. during long - term chronoamperometric experiments (1 h), there was only 15 % decrease of catalytic currents in the case of tungsten oxides. in both experiments, no significant deactivation effect was observed that may imply dissolution of wo3. in order to gather information of the ability of coads poisoning species to undergo oxidative desorption from the surface of the prepared pt - based catalysts, co - stripping voltammetry was performed. a typical coads - stripping curve on bare ptir / vulcan catalyst is presented in fig. it is characterized by a single sharp and prominent coads oxidation peak centered at 0.69 v with a coads oxidation onset potential of 0.590 v. in contrast, for the wo3-modified ptir / vulcan catalysts, two separated coads oxidation peaks with the position of the main peak potential at 0.62 v were found in the stripping voltammetric method (fig . the onset potential for the main coads oxidation peak starts near 0.49 v. it becomes broadened and shifts to a more negative potential ( ca . 100 mv) versus the main coads oxidation peak for the bare ptir / vulcan. for both catalysts, the hydrogen region was completely blocked by the full coverage with coads; the main co - stripping (oxidation) peak appeared only during the first anodic cyclic, which indicates that all adsorbed co was oxidized and removed from the surface under this condition. this observation is in good agreement with the previous reports for metal oxides (e.g., wo3, moo3).fig. 7co - stripping voltammograms recorded at 10 mv s in 0.5 mol dm h2so4 for the ptir / vulcan (a) and wo3-modified ptir / vulcan (b) catalysts. co adsorption was done at 0.1 v. solid curve shows the first cycles, while the dot curve shows the second cycles co - stripping voltammograms recorded at 10 mv s in 0.5 mol dm h2so4 for the ptir / vulcan (a) and wo3-modified ptir / vulcan (b) catalysts. co adsorption was done at 0.1 v. solid curve shows the first cycles, while the dot curve shows the second cycles the presence of two peaks on the co - stripping voltammetry has been described in the literature. two signals observed for oxidation of co - adsorbed curve on the wo3-modified ptir / vulcan catalyst suggest that two active sites for coads oxidation may exist, which are the likely case due to a variation in the interaction with the metal oxide. in a previous report, this effect for pt - based electrode has been explained by the presence of at least two different types of co adsorption products (linearly or bridged bound) which are characterized by different binding energies. moreover, the peak that appears at the lower potential may be ascribed the coads oxidation on the surface of wo3-modified ptir / vulcan catalyst corresponding to the interaction between the pt nanoparticles and tungsten oxide, whereas the peak at the higher potential may be attributed to adsorption on pt nanoparticles at a large distance from tungsten oxide. additionally, it can not be excluded that this peak originates from the redox reaction of a tungsten species. to estimate the electrochemically active surface area (sa), the hydrogen adsorption / desorption system can not be used because of the overlap with the redox process of tungsten oxide. the co - stripping voltammetry method relies on forming a monolayer of strongly adsorbed co on the bare and modified ptir / vulcan catalysts. generally, co may form a linear, bridge bond to the surface leading to different numbers of electrons per site (2 and 1, respectively). a linear adsorption may dominate if the co adsorption occurs at a potential close to 0 v. by analogy to these conditions, it was assumed that one monolayer of co adsorbed on pt (linked linearly) and that the coulombic charge required to oxidize adsorbed co to co2 is equal to 420 c cm. the charge value required to estimate the electrochemically active surface area was determined by integrating the main co - stripping peaks. the obtained sa values for the catalysts were 57 and 54 m g for bare ptir / vulcan and wo3-modified ptir/ vulcan, respectively. it is reasonable to conclude that the electrochemical active surface area is not the major factor causing the difference of their catalytic activities for ethylene glycol and for ethanol electrooxidations. in other words, tungsten oxide species occupied slightly the electrochemically active surface area of modified ptir / vulcan nanoparticles. the same tendency has been noticed by others with different catalysts (e.g., pt, ptsn, and ptrh) and with various transition metal oxides. these effects may be explained by the ability of tungsten oxide in contact with aqueous solutions to increase the population of surface hydroxyl groups which likely play a major role in the coads removal. further work is needed to determine whether such catalysts are stable under test conditions for fuel cells to elucidate full reaction pathways. herein, we demonstrate the enhancement of the activity of catalysts composed of bimetallic ptir nanoparticles and tungsten oxide toward electrooxidation of ethylene glycol and ethanol. from both xrd and tem , the average particle sizes were found to be in the range of 48 nm and uniformly dispersed on glassy carbon. the adsorbed layer of tungsten oxide on ptir / vulcan nanoparticles increases the catalytic currents and decreases the onset potentials for electrooxidation of ethylene glycol and ethanol. the above electrochemical measurements confirmed that the presence of tungsten oxide on the surface is beneficial in the electrooxidation of ethylene glycol and ethanol. the also showed that the activity of the ptir / vulcan nanoparticles for oxidation of poisoning species (coads) is higher in the presence of tungsten oxide. the activation effect may involve direct specific interactions (chemical or electronic) between wo3 and both pt and ir metals.

in this article, we characterized tungsten oxide - decorated carbon - supported ptir nanoparticles and tested it for the electrooxidation reactions of ethylene glycol and ethanol. phase and morphological evaluation of the proposed electrocatalytic materials are investigated employing various characterization techniques including x - ray diffraction (xrd) and transmission electron microscopy (tem). electrochemical diagnostic measurements such as cyclic voltammetry, chronoamperometry, and linear sweep voltammetry revealed that the tungsten oxide - modified ptir / vulcan nanoparticles have higher catalytic activity for ethylene glycol and ethanol electrooxidation than that of ptir / vulcan. a significant enhancement for electrooxidation of co - adsorbate monolayers occurred in the presence of a transition metal oxide relative to that of pure ptir / vulcan electrocatalyst. the likely reasons for this are modification on the pt center electronic structure and/or increasing the population of reactive oxo groups at the ptir / vulcan electrocatalytic interface in different potential regions.

the use of invasive mechanical ventilation by endotracheal intubation in acute respiratory failure is long known.1 noninvasive ventilation (niv) began to be considered as an alternative to invasive mechanical ventilation in acute respiratory failure caused from exacerbations of chronic obstructive pulmonary disease (copd) in the 1980s, and its use gradually rose worldwide. in 1998, an international prospective survey reported that about one - third of patients initially treated with niv had to undergo endotracheal intubation,2 but 6 years later another large international prospective study reported a better outcome.3 also, niv effectiveness is supported by of systematic reviews,4 and this has led to an increase in the application of this treatment, even in complicated situations such as a do - not - intubate order in elderly patients.5 however, in 2010, manuel et al reasoned that there is little convincing evidence for the use of niv in severe, but stable copd and that what is less clear, however, is the quality of how niv is delivered to patients.6 indeed, a recent study focused its attention on the kind of niv setup, which can be based on high inflation pressure and high backup rate (high - intensity niv) or high pressure and low backup rate (high - pressure niv). the authors reported that the pivotal role in managing by niv hypercapnic respiratory failure in copd patients is the high - pressure component.7 seventy - eight patients (57 males, mean age 78.3 9.2 years) undergoing niv were evaluated. of them, 48 (62.3%) had acute hypercapnic respiratory failure because of a copd exacerbation and the remaining 30 had acute hypercapnic respiratory failure from other causes, mainly cardiac failure. all patients were treated by niv using the bi - level positive airway pressure set up at high pressure / high backup rate. niv was successful in 67 subjects (85.9%) and the patients were discharged, 57 of whom continued niv at home and ten were in a state of spontaneous breathing. niv was unsuccessful in eleven patients, ten of whom died and one was successfully treated by invasive mechanical ventilation. the mean age of successfully treated (77.7 9.4 years) and unsuccessfully treated (82.1 8.1 years) patients was not significantly different. significant differences were detected for a higher basal glasgow coma scale score in successfully treated patients (p = 0.007), a higher basal score of the acute physiology and chronic health evaluation score in unsuccessfully treated patients (p = 0.004), and a lower ph after 1 hour in unsuccessfully treated patients (p = 0.015). among the risk factors for failure of niv, the ph value seems particularly important,8 which corresponds with the current data. also, the acute physiology and chronic health evaluation confirmed the predictive value of a score higher than 29, which was detected in patients with a negative outcome. overall, the current data, obtained in a pneumology unit, show that niv is able to manage the large majority of patients (86%) with acute respiratory failure from copd exacerbations and also from other causes. a randomized study found that the use of niv as rescue therapy was associated with a lower number of patients meeting the endotracheal intubation criteria and with a lower mortality rate compared with endotracheal intubation.9 the factors underlying a negative outcome of niv warrant investigation in studies in large populations of patients. a recent study reported that patients with copd and obesity hypoventilation syndrome treated with niv because of acute hypercapnic respiratory failure had a response to treatment similar to patients with only copd.10 moreover, a low rate of endotracheal intubation need was recently reported in patients with acute respiratory failure from heart failure treated with niv,11 and this observation is pertinent for the patients in the current study. the findings show a very high rate of success of niv in patients with acute hypercapnic respiratory failure from copd as well as from other causes, including cardiac failure. this suggests that the use of invasive mechanical ventilation may be further reduced, with a decrease in its known complications as well.

noninvasive ventilation (niv) was introduced as an alternative to invasive mechanical ventilation for acute respiratory failure caused from exacerbations of chronic obstructive pulmonary disease in the 1980s, and its use gradually rose worldwide. seventy - eight patients (57 males, mean age 78.3 9.2 years) undergoing niv were evaluated. of them, 48 (62.3%) had acute hypercapnic respiratory failure because of a chronic obstructive pulmonary disease exacerbation, and the remaining 30 had acute hypercapnic respiratory failure from other causes, mainly cardiac failure. all patients were treated by niv using the bi - level positive airway pressure set up at high pressure / high backup rate. niv was successful in 67 subjects (85.9%) and the patients were discharged, 57 of whom continued niv at home and ten had spontaneous breathing. niv was unsuccessful in eleven patients, ten of whom died and one was successfully treated by invasive mechanical ventilation. significant differences were detected for a higher basal glasgow coma scale score in successfully treated patients (p = 0.007), a higher basal acute physiology and chronic health evaluation score in unsuccessfully treated patients (p = 0.004), and a lower ph after 1 hour in unsuccessfully treated patients (p = 0.015). these findings show a very high rate of success of niv in patients with acute hypercapnic respiratory failure not only from chronic obstructive pulmonary disease but also from cardiac failure. this suggests that the use of invasive mechanical ventilation may be further reduced, with a decrease in its known complications as well.

neuromyelitis optica (nmo) and multiple sclerosis (ms) are autoimmune inflammatory demyelinating disorders of the central nervous system with unknown etiology, causing nontraumatic neurological disability in young adults. nmo is thought to be a subtype of ms but not an independent disease until the discovery of the anti - aquaporin 4 (aqp4) antibody or nmo - igg. although the exact etiologies of both diseases are still unclear, it is sure that the t - helper cells (th), directly or indirectly, participate in the pathogenesis and progress of ms and nmo by secreting various cytokines. over recent years, interleukin-7 (il-7) had been widely considered to be a key cytokine controlling the differentiations and immune responses of several t - cells subsets. it is suggested that the function of il-7 was not only stimulating il-7 receptor alpha (il-7r) to promote the differentiation of th1, but also involving in the survival and proliferation of pathogenic th17 cells in experimental autoimmune encephalomyelitis (eae) and ms patients. the serum il-7 can be seen as a potential marker monitoring the response to interferon- in ms patients. the genetic variants of il-7 and il-7ra had been identified to be associated with the susceptibility of ms and nmo in caucasian, japanese and some other populations in several studies. however, the had not been replicated in chinese han population except our previous study on rs1520333 in il-7 and rs6897932 in il-7ra, in which no positive associations were observed. as the il-7/il-7r pathway might be a very attractive therapeutic target for inflammatory disorders, the genetic variants in this pathway were proposed to implicate in the pathogenesis of various autoimmune diseases. here, we enlarged the sample size to replicate the association of these 2 single nucleotide polymorphisms (snps) (rs1520333 in il-7 and rs6897932 in il-7ra) with ms and nmo. meanwhile, we assayed 5 more snps of il-7 (rs1545298, rs4739140, rs6993386, rs7816065, and 2887502) in the enlarged samples of ms, nmo, and healthy controls. as described in our previous study, 110 unrelated nmo patients and 304 healthy controls were included. in this study, we recruited 57 more nmo patients and 178 more healthy controls. in total, 167 nmo patients (26 males and 141 females) and 479 healthy controls (255 males and 224 females) among chinese han population in southeastern china were included in this study. in addition, we recruited 159 ms patients (66 males and 93 females), and the diagnosis of which met the 2005 mcdonald criteria for ms. nmo - igg antibodies were detected with an indirect immunofluorescence assay using human embryonic kidney 293 cells transfected with recombinant human aqp4 gene (each sample was measured at least twice, with the examiners unknowing the origin of the specimens . genomic dna was extracted from peripheral blood samples using a qiaamp dna blood minikit ( qiagen, hilden, germany). six variants in il-7 (rs1520333, rs1545298, rs4739140, rs6993386, rs7816065, and 2887502) and one variant in il-7ra (rs6897932) were genotyped using sequenom massarray system (sequenom, san diego, ca, usa) in previous 106 nmo patients and 304 healthy controls, according to the manufacturer's instructions at fudan - van andel research institute center, as we previously described. for quality control, sample duplicates whose genotypes had been identified by sequencing were included in each 96-well plate. then, the additional samples including 57 nmo patients, 159 ms patients, and 178 healthy controls were genotyped by sanger sequencing using an abi 3730 automated dna sequencer (applied biosystems, usa). primer sequences for analysis of il7 and il7ra variants pcr: polymerase chain reaction. hardy the genotypes and allele frequencies were compared between cases and controls using the chi - square test or fisher's exact test. continuous variables were shown as mean standard deviation (sd). a p < 0.05 was considered as statistically significant. as described in our previous study, 110 unrelated nmo patients and 304 healthy controls were included. in this study, we recruited 57 more nmo patients and 178 more healthy controls. in total, 167 nmo patients (26 males and 141 females) and 479 healthy controls (255 males and 224 females) among chinese han population in southeastern china were included in this study. in addition, we recruited 159 ms patients (66 males and 93 females), and the diagnosis of which met the 2005 mcdonald criteria for ms. nmo - igg antibodies were detected with an indirect immunofluorescence assay using human embryonic kidney 293 cells transfected with recombinant human aqp4 gene (euroimmun, lubeck, germany). each sample was measured at least twice, with the examiners unknowing the origin of the specimens. samples with twice positive were reported to be nmo - igg positive. genomic dna was extracted from peripheral blood samples using a qiaamp dna blood minikit (qiagen, hilden, germany). six variants in il-7 (rs1520333, rs1545298, rs4739140, rs6993386, rs7816065, and 2887502) and one variant in il-7ra (rs6897932) were genotyped using sequenom massarray system (sequenom, san diego, ca, usa) in previous 106 nmo patients and 304 healthy controls, according to the manufacturer's instructions at fudan - van andel research institute center, as we previously described. for quality control, sample duplicates whose genotypes had been identified by sequencing were included in each 96-well plate. then, the additional samples including 57 nmo patients, 159 ms patients, and 178 healthy controls were genotyped by sanger sequencing using an abi 3730 automated dna sequencer (applied biosystems, usa). hardy weinberg equilibrium (hwe) was tested using the chi - square test. the genotypes and allele frequencies were compared between cases and controls using the chi - square test or fisher's exact test. continuous variables were shown as mean standard deviation (sd). a p < 0.05 was considered as statistically significant. all data were analyzed using spss 16.0 software for windows (spss inc ., chicago, il, usa). after genotyping by sequenom massarray system, 4 nmo patients and 14 healthy controls were excluded for genotyping success rate < 90%. hence, there were 163 nmo patients, 159 ms patients, and 468 healthy controls analyzed. nmo - igg antibody was tested in the 57 newly recruited nmo patients, and 13 (29.5%) were positive, added with previous 44 antibody positive nmo patients, there were 57 nmo - igg antibody positive patients in total. most of the snps in each group were under the hwe except il-7 rs2887502 in ms (p = 0.033) and il-7ra rs6897932 in healthy controls (p = 0.031). for the c / c genotype of il-7 rs2887502 did not exist in ms patients and the frequency of c / c genotype was much higher in healthy control than that in the nmo - igg positive nmo patients (70.30% vs. 61.40%), the departure from dhw of both polymorphisms in ms and healthy controls were possibly due to the protective role of c / c genotype according to a previous study. characteristics of all chinese han participants in this study ms: multiple sclerosis; nmo: neuromyelitis optica; aqp4-ab: antiaquaporin-4 antibodies; na: not applicable; sd: standard deviation. the upper and the bottom panels indicate the homozygous genotypes, whereas the heterozygous genotype is shown in the middle one. hardy weinberg equilibrium tests for all chinese han participants in this study a p < 0.05 was considered statistically significant. ms: multiple sclerosis; nmo: neuromyelitis optica; il7: interleukin-7; il7ra: interleukin-7 receptor alpha. as listed in table 4, after enlarging the sample size for both previously studied snps, the frequency of the a / a genotype of rs1520333 in il-7 gene of ms patients was observed dramatically lower than healthy controls, which reached the statistical difference (p = 0.035). meanwhile, although statistical difference of the genotype was existed between total nmo and healthy controls in il-7ra rs6897932 (p = 0.034), we also found no statistical difference in the allele frequencies (p > 0.05). when comparisons were made between nmo - igg positive patients and ms patients or healthy control, we found that statistical significant differences of genotype and allele distributions were observed in il-7ra rs6897932 (p = 0.004 and p = 0.042) between the nmo - igg positive patients and healthy controls. allele and genotype distributions of il7 and il7r variants among ms, nmo and healthy controls, n (%) * the of fisher's exact test. ms: multiple sclerosis; nmo: neuromyelitis optica; nmo: total neuromyelitis optica patients; nmo: neuromyelitis optica - igg positive patients; il7: interleukin-7; il7ra: interleukin-7 receptor alpha. the of five newly assayed il-7 snps are listed in table 5, along with the of corresponding chi - square test. the genotypes of il-7 rs2887502 were significantly different between nmo and ms patients (p = 0.014). and allele and genotype distributions of 5 newly studied il7 variants among ms, nmo and healthy controls, n (%) * the of fisher's exact test. ms: multiple sclerosis; nmo: neuromyelitis optica; nmo: total neuromyelitis optica patients. autoimmune diseases are complex trait that develop from intricate and poorly understood interactions between an individual's genetics and the environmental exposures. the genetics of nmo susceptibility largely remain unknown. in this study, we totally investigated the association of 6 variants in il-7 and one variant in il-7ra with chinese nmo and ms patients, especially with the nmo - igg positive patients. we observed that the genotype of rs6897932 in il-7ra was statistically different between nmo patients and the healthy controls after including more nmo patients and healthy controls. while further comparing the nmo - igg positive patients with healthy controls, we found that the c / c genotype and c allele significantly decreased in nmo - igg positive nmo patients. in il-7 gene, the genotypes of il-7 rs2887502 were observed statistically different between nmo and ms patients in our present study. il-7 belongs to a superfamily of gamma - chain cytokine receptor, and its gene, il-7, is located on chromosome 8q12 - 13. functionally, il-7 binds to il-7r (also termed as cd127), which is encoded by the gene il-7ra, plays an essential role in the t cell survival and proliferation in human and animal model. a recent study revealed that the serum il-7 reflected the ms patients responsiveness to interferon- in th1-dirven ms, because of the important role of il-7 played in enhancing the th1 proliferation. in turn, numbers of previous studies pointed out the il-7/il-7ra variants were associated with the morbidity of nmo and ms in different populations and regions of the world. the rs6997932 was proved to be a causative variant that affected the expression of il-7ra. studies on different populations reached a consensus that this snp was strongly associated with ms, moreover, it was also associated with nmo in the studies performed in japanese and korean populations. some studies revealed that rs6993386, rs1520333, and rs7816065 of il-7 were associated with ms in caucasian populations, while rs1545298, rs4739140, and rs2887502 did not relate to this disease. previously, we performed a partial replication of the referred studies by assayed rs1520333 of il-7 and rs6897932 of il-7ra, however, we did not find any positive association of both snps with nmo. in consideration of the different genetic among populations , we enlarged the sample size and investigated the associations of more snps with not only nmo patients, but also ms patients in the current study. interestingly , statistical significant differences were observed in the genotypes of il-7ra rs6897932 between the cohorts of total nmo patients and healthy controls. while further comparing the nmo - igg positive patients with healthy controls, significant differences existed in both distributions of genotypes and alleles in il-7ra rs6897932. this was coherent with the of studies in japanese and korean populations, confirming that il-7ra rs6897932 might be a risky factor of nmo in asians. however, we did not observe the association of il-7ra rs6897932 with the pathogenesis of ms, which indicated the different genetic among the east asians. in the comparisons between the ms patients and healthy controls, the genotypes of il-7 rs1520333 reached the statistical significant difference, which was first reported in asian. the significant difference of il-7 rs2887502 genotypes between nmo and ms patients was also firstly reported, which indicated the different genetic s of both diseases. although our study reported a potential association between il-7/il-7ra polymorphisms and ms / nmo, some limitations were present and should be addressed in the future. second, there was a disparity in the gender ratio, since the incidences of both diseases are higher in female than male. lastly, functional studies of the il-7 would be required to understand the actual effect of the il-7/il-7r complex in ms and nmo pathogenesis. in , the current study replicated some positive - associated snps, which were observed in other populations, in chinese nmo and ms patients. meanwhile, we investigated the associations of 5 more snps of il-7 with nmo and ms that were not investigated in asian previously. to our best knowledge, this study may provide a deeper understanding of the role of il-7/il-7r pathway playing in the pathogenesis of nmo and ms within different populations, which hints a new insight to the personal therapy for both diseases in the future. and this study suggested that it might be necessary to monitor the serum il-7 of nmo - igg positive patients during the treatment. this work was supported by grants from national key clinical specialty discipline construction program and key clinical specialty discipline construction program of fujian and the national natural science foundation of china ( no. 81125009 and no. this work was supported by grants from national key clinical specialty discipline construction program and key clinical specialty discipline construction program of fujian and the national natural science foundation of china ( no. 81125009 and no.

: neuromyelitis optica (nmo) and multiple sclerosis (ms) are autoimmune demyelinating diseases of the central nerve system. interleukin-7 (il-7) and interleukin-7 receptor alpha (il-7r) were proved to be important in the pathogenesis of both diseases because of the roles they played in the differentiations of autoimmune lymphocytes. the variants of both genes had been identified to be associated with ms susceptibility in caucasian, japanese and korean populations. however, the association of these variants with nmo and ms has not been well studied in chinese southeastern han population. here , we aimed to evaluate the association of six il-7 variants (rs1520333, rs1545298, rs4739140, rs6993386, rs7816065, and rs2887502) and one variant of il-7ra (rs6897932) with nmo and ms among chinese han population in southeastern china.methods:matrix-assisted laser desorption / ionization time of flight mass spectrometry (massarray system) and sanger sequencing were used to determine the variants of il-7 and il-7ra in 167 nmo patients, 159 ms patients and 479 healthy controls among chinese han population in southeastern china. samples were excluded if the genotyping success rate < 90%.: statistical differences were observed in the genotypes of il-7 rs1520333 in ms patients and il-7ra rs6897932 in nmo patients, compared with healthy controls (p = 0.035 and 0.034, respectively). there was a statistically significant difference in the genotypes of il-7 rs2887502 between ms and nmo patients (p = 0.014). and there were statistically significant differences in the rs6897932 genotypes (p = 0.004) and alleles (p = 0.042) between nmo - igg positive patients and healthy controls.:the study suggested that among chinese han population in southeastern china, the variant of il-7ra (rs6897932) was associated with nmo especially nmo - igg positive patients while the variant of il-7 (rs1520333) with ms patients. and the genotypic differences of il-7 rs2887502 between ms and nmo indicated the different genetic s of these two diseases.

flavonoids comprise a large group of plant metabolites, 6,000 of which have been identified to date. quercetin is a major natural flavonoid existing in the skin of many fruits and vegetables, as well as in leafy vegetables, berries, black tea, and various fruit juices. since it can be found in many plant - derived foods, it is present in most people's diet. having many antioxidant, anticarcinogenic, anti - inflammatory, and heart - protecting effects, quercetin decreases the risk of cancers and various chronic diseases. thus, it is produced as a dietary supplement and is even added to some food and beverages. despite the large number of studies investigating the in vitro effects of quercetin, few have demonstrated its in vivo effects. there might be differences between the in vivo and in vitro effects of quercetin owing to its digestion, absorption, and metabolism effects. some animal studies have described the observed increased endurance performance and maximal oxygen consumption (vo2max) and therefore fitness following quercetin consumption as a of elevated number of intracellular mitochondria caused by the flavonoid. however, this association has not yet been proved in human studies. the improved physical performance caused by quercetin reported by some studies might be attributable to the decreased membranes in muscles, which in turn reduces the negative and exhaustive effects of excessive oxygen radicals during physical activity. proving this hypothesis to be right would mean quercetin to be able to reduce muscular damage and soreness, as well as neuromuscular dysfunction following exercise. however, quercetin has been reported to have contrasting antioxidant effects. although some studies found improved neuromuscular function and decreased soreness, others only mentioned improved muscular strength as a of long - term quercetin consumption. however, they did not find quercetin to have significant effects on these indices. as mentioned previously, although in vitro and animal studies have suggested quercetin to have positive effects on athletic performance, inflammatory indices, and muscular damage, a definite judgment can not be made about its effects on athletes because of the fewer number of human studies and their contrasting findings. on the other hand, there is a growing interest in supplement use among athletes. therefore, this study evaluated the effects of supplementary quercetin on athletic performance, muscular damage, and body composition in male athlete students. this double - blind clinical trial involved 60 male students at isfahan university of medical sciences having an athletic history of at least 3 years. the subjects were excluded if they followed less than 70% of the study procedure or were unwilling to continue. the trial was approved by the research and ethics committee, faculty of nutrition, isfahan university of medical sciences, isfahan, iran, and registered in irct. the participants were first explained about the objectives and methods of the study. after obtaining written informed consents, height was measured to the nearest 0.5 cm by a tape meter in standing position without shoes. weight was measured in light clothing without shoes by a scale with an accuracy of 100 g, following which body mass index (bmi) was calculated as weight divided by squared height (kg / m). a bioimpedence device was used to measure body water percentage and fat - free mass percentage. to evaluate body performance indices, exercise test was performed for all participants using the bruce protocol and hp cosmos treadmill (mercury, germany). at the end of the test, vo2max and the individuals were then assigned into four groups of 15 using permuted block randomization. the first to fourth groups received a 500 mg supplemental quercetin capsule plus a 250 mg vitamin c pill, a 500 mg supplemental quercetin capsule plus a 250 mg placebo vitamin c pill, a 500 mg placebo quercetin capsule plus a 250 mg vitamin c pill, and a 500 mg placebo quercetin capsule plus a 250 mg placebo vitamin c pill, respectively, daily for 8 weeks. the participants were asked to continue their routine diet and physical activity during the study and they were monitored through phone calls or text messages. at the end of the intervention period, the remainder of the pills was evaluated to assess supplement intake. due to the double - blind nature of the study, neither the researchers nor the participants were aware of the content of the capsules. data were analyzed by paired t - test, analysis of covariance (ancova), and repeated measure analysis of variance (anova) in spss15. data were analyzed by paired t - test, analysis of covariance (ancova), and repeated measure analysis of variance (anova) in spss15. since four individuals were excluded due to unwillingness or other reasons, a total number of 56 participants were studied. mean ages of subjects in groups 1 to 4 were 20.93 1.53, 21.50 2.17, 21.21 1.52, and 20.46 1.18 years, respectively (p > 0.05). table 1 shows the body composition indices among the four groups before and after the study. as seen, lean body mass (lbm), total body water (tbw), basal metabolic rate (bmr), and total energy expenditure (tee) increased significantly in the first group. the comparison of body composition indices between four groups as presented in table 2, body performance indices shows vo2max in group 1 significantly increased after the intervention. in addition, as the of ancova after excluding the initial effects of vo2max suggested, vo2max increased in the first and third groups following the intervention. however, repeated measure anova did not reveal the differences to be significant. although the distance covered significantly increased in group 4, ancova and repeated measure anova did not show significant differences. this randomized clinical trial comprised four groups who were evaluated to determine the effects of quercetin intake on physical and body performance and muscle injury. the revealed significant differences in lactate dehydrogenase (ldh), vo2max, tee, tbw, and lbm among the quercetin and vitamin c groups. however, after eliminating confounding effects of initial variables, only vo2max changes remained significant. our study is in line with the study of cureton et al., who assessed the ergogenic effects of quercetin on 30 non - athletic men in a double - blind clinical trial. during 7 to 16 days, they administered 1 g of daily quercetin to the intervention group and the same amount of placebo to the control group. finally, they did not find significant changes either in ergogenic indices, such as phosphocreatine recovery time constant, vo2max, and perception of effort, during submaximal exercise test, or in the total work during a 10-min cycling with maximum power. similarly, ganio et al. performed a double - blind clinical trial on 11 non - athletic inactive participants (five males and six females). although their intervention and control groups received, respectively, 1,000 mg of daily quercetin and placebo for 22 weeks, no significant differences were observed in terms of vo2max between the two groups. utter et al. also compared the effects of 250 mg of daily quercetin for 3 weeks with placebo among marathon runners and cyclists. they did not report any significant difference in the ratings of perceived exertion between the two groups. a crossover clinical trial by macrae et al. compared the effects of quercetin and a combination of quercetin and vitamins on 11 male cyclists during a 6 week period. although their did not reveal significant differences in the total time of a 30 km ride and vo2max after the intervention, a significant increase was observed in peak power among the second group of cyclists. in a clinical trial, davis et al. evaluated the effects of quercetin on exercise performance among 12 volunteers. they divided the participants into two groups of intervention (500 mg daily quercetin) and control (500 mg daily placebo). unlike in other studies, they found significant improvements in time to fatigue, vo2max, and endurance among non - athletic individuals during a 30 km bicycle ride. they performed a 12 week treatment with two doses of quercetin along with vitamin c and niacin on 941 male and female subjects aged 18 - 85 years. the participants were randomly divided into three groups of 500 mg daily quercetin, 1,000 mg daily quercetin, and placebo. their did not show any significant differences between the intervention and placebo groups in terms of bmi or any other body composition indices. egert et al. investigated the effects of quercetin consumption on 93 obese subjects aged 25 - 65 in a crossover study. the participants received 6 weeks of 150 mg daily quercetin followed by a 5 week washout and a course of placebo. however, crp and body composition indices, including weight, waist circumference, body fat mass, and fat - free mass, did not significantly change after the intervention period. another study by egert et al. evaluated 35 healthy subjects in three groups of 50, 100, and 150 mg of daily quercetin. the study did not indicate any significant difference in resting energy expenditure or weight after a 2 week quercetin supplementation. similar to previous studies, the present study did not show any significant differences between the quercetin and placebo groups. therefore, it might be concluded that quercetin alone does not affect body composition and body performance indices. although different studies have used various doses of quercetin during various periods of time, we selected a dose within their range. however, higher doses might be able to make significant differences in the above - mentioned indices. since the findings of the present and previous studies are in contrast with in vitro and animal studies, a longitudinal research with long follow - up is suggested to evaluate the effects of quercetin. moreover, studies assessing the effects of quercetin on cardiovascular disease risk factors, endothelial dysfunction, and atherosclerosis incidence might reveal beneficial effects of this flavonoid. clinical trials with larger sample sizes of athletes and non - athletes are also recommended.

: flavonoids comprise a large group of plant metabolites, 6,000 of which have been identified to date. some studies have shown the increased aerobic performance and maximal oxygen consumption (vo2max) and therefore fitness following quercetin intake as a of elevated number of intracellular mitochondria caused by the flavonoid.methods:this double - blind clinical trial comprised 60 male students having an athletic history of at least 3 years. body composition, exercise performance, and some blood biomarkers were analyzed. the individuals were selected by convenient sampling, and then were assigned into four groups of equal number by using permuted block randomization. the first to fourth groups received a 500 mg supplemental quercetin capsule plus a 250 mg vitamin c pill, a 500 mg supplemental quercetin capsule plus a 250 mg placebo vitamin c pill, a 500 mg placebo quercetin capsule plus a 250 mg vitamin c pill, and a 500 mg placebo quercetin capsule plus a 250 mg placebo vitamin c pill, respectively, daily for 8 weeks. the participants were asked to continue their routine diet and physical activity during the study and they were monitored through phone calls or text messages.:lean body mass, total body water, basal metabolic rate, and total energy expenditure increased significantly in the quercetin group after intervention. on the other hand, vo2max increased in the quercetin and quercetin + vitamin c groups following the intervention, non-significantly.:our findings suggest that supplementation with quercetin in athletes may improve some indices of performance.

the modified mallampati classification (mmp) is widely used for pre - operative assessment of airway, and mallampati scores of iii and iv have been reported to be valuable in predicting difficult laryngoscopy in acromegalic patients as well. as it is known that mmp not only assesses pharyngeal structures but also head and neck mobility. it has been suggested that craniocervical extension relates to mouth opening and limited head and neck mobility can in poor mmp. mashour and sandberg found that application of craniocervical extension improves the specificity and positive predictive value while retaining the sensitivity of the traditional mmp examination in a group of normal patients. the addition of craniocervical extension to the mmp was referred as the extended mallampati score (ems). the incidence of difficult intubation in acromegalic patients is higher than the general surgical population. there are numerous reports of difficult laryngoscopy and intubation in patients with acromegaly. to date, literature search did not reveal studies assessing the application of ems in for predicting difficult intubation in acromegalics. the primary aim of this study was to compare ems with mmp in predicting difficult laryngoscopy in acromegalic patients. we hypothesized that since ems has been reported to be more specific and better predictor than mmp, it may be superior to the mmp to predict the incidence of difficult laryngoscopy in acromegalic patients. our local ethics committee approved this prospective, observational, and controlled study and a written informed consent was obtained from all patients. over a period of 3 years (january 2008-december 2010) 39 consecutive acromegalic patients presenting for for excision of pituitary tumor were enrolled. acromegalic features such as prognathism, macroglossia, and soft - tissue swelling were present variably in acromegalic group. for each acromegalic patient enrolled, the subsequent non - acromegalic patient with pituitary tumor was also enrolled to serve as a control. pre - operative airway assessment was performed by two experienced anesthesiologists and involved a mmp with patient sitting with the head in neutral position without phonation and then the ems with head in full extension, as described in literature. the view on mmp and ems was graded as follows: class i = soft palate, fauces, uvula, and pillars visible; class ii = soft palate, fauces, and uvula visible; class iii = soft palate and base of the uvula visible; class iv = soft palate not visible at all. airway management plan for each patient was left to the discretion of the attending anesthesiologist who performed independent pre - operative assessment. standard monitoring prior to induction included electrocardiogram, non - invasive blood pressure, and pulse oximetry. after pre - oxygenation, general anesthesia was induced with fentanyl (2 mcg / kg) and propofol (1.5 - 2 mg / kg). oropharyngeal airway and/or two - hand mask hold was used to facilitate mask ventilation, when required. after loss of all four twitches on the train - of - four obtained by ulnar nerve stimulation at the wrist, laryngoscopy was performed by one of the two attending anesthesiologists, using an appropriately sized macintosh laryngoscope blade and the laryngeal view was classified according to the method of cormack and lehane (grade i = full view of the glottis, grade ii = glottis partly exposed, anterior commissure not seen, grade iii = only epiglottis seen, or grade iv = epiglottis not seen). however, it was allowed thereafter on instruction of the laryngoscopist in patients with initial laryngeal view grades iii and iv. both, mmp and ems grades iii and iv were considered predictors of difficult laryngoscopy, which was defined as cormack - lehane (cl) grades iii or iv (before external laryngeal manipulation elm). intubation was defined as difficult if it required more than two attempts, change of blade, or use of a gum - elastic bougie / fiber optic bronchoscope / intubating laryngeal mask airway. the pre - operative assessment data were used to determine the accuracy of the two tests in predicting difficult intubation. several statistical measures were calculated that have been frequently used and are provided in appendix 1. statistical analysis was done using software stata 9.1 (college station, texas, usa). data are presented as mean (standard deviation, sd), number or percentage. the demographic data were compared using two sample t - test with equal variance. the value of p < 0.05 was considered significant. a total of 78 patients participated in the study and one patient from control group was excluded from final analysis. in the control group, 39 patients were enrolled and one was excluded from final calculations because of inability to obtain cl grade. in the acromegalic group, a total of eight patients in acromegalic group and six patients in control group had a difficult airway manifesting as grade iii and iv on laryngoscopy without external laryngeal manipulations. demographic deatils of the study groups mean (sd) or n relationship between modified mallampati test and extended mallampati score with cormack - lehane grading of laryngoscopic view in the two study groups incidence of class iii, iv mmp assessment in normal and extended position was 18% and 31% in acromegalics. the sensitivity of both the positions was equal (13%) whereas specificity for mmp and ems was 81% and 65% respectively in acromegalics. almost similar values of negative predictive value were obtained (78% for mmp and 74% for ems) on contrary mmp was associated with higher positive predictive value (14%) as compared to ems (1%). the incidence of class iii, iv mmp assessment in normal and extended position was 13% for both positions in control group. positive predictive value for mmp and ems was 60% and 0% respectively, whereas negative predictive value associated with these positions was 91% and 82%. statistical terms used for modified mallampati test and extended mallampati score as predicting tests for difficult laryngoscopy (number or %) the incidence of difficult intubation in general population has been described to be 5.8% in overall population, 6.2% for normal patients excluding obstetric and obese patients, 3.1% for obstetric patients and15.8% for obese patients by shiga et al. in meta - analysis of bedside screening test performance. mallampati test has been performed in with and without phonation and/or with different head or tongue positions in these studies, but despite theoretical arguments, this test has yielded poor to moderate sensitivity and moderate to fair specificity (moderate discriminative power). pooled sensitivity of mmp is 49% and specificity being 86%, positive likelihood ratio 3.7, and negative likelihood ratio 0.5. diagnostic efficacy of these screening tests has been reported variously because of multiple factors like different patient characteristics, different test thresholds, inadequate statistical power and variable and low incidence of difficult intubation. incidence of class iii, iv mmp assessment in normal and extended position was 13% for both positions in control group. heterogeneity in mallampatti data has been reported with likely reasons being inconsistency and uncertainty in performing these tests. the incidence of difficult intubations in acromegalics in two retrospective studies has been described as 12 and 30/100 patients. in a study by schmidt et al. they defined difficult intubation as greater than two attempts at intubation, use of gum elastic bougie or change of laryngoscope blade. they reported 26% incidence of difficult laryngoscopy grade (cl iii) but with application of elm, it decreased to 10%. none of their patients had cl iv laryngoscopy grade. in our study, the incidence of class iii, incidence of difficult laryngoscopy as assessed by cl grade (iii, iv) was 20% in our study. the discrepancy may be because of the degree of acromegaly which may have led to more difficult airway. multiple bed side tests have been proposed for anticipation of difficult laryngoscopy and intubation, most common being the mmp. recently, the upper lip bite test has also been used and compared with mmp to predict difficult laryngoscopy in acromegalic patients. so far these tests have reported poor to moderate (20 - 62%) sensitivity with moderate to fair specificity (82 - 97%) as described by shiga et al. in their meta - analysis of 35 studies enrolling 50,760 patients. the diagnostic accuracy of these tests varies from trial to trial because of inter - observer differences, different test thresholds, inadequate statistical power and racial variation. the pooled sensitivity of mmp has been described 41 - 57%, pooled specificity is 81 - 90%. difficult intubation has been described variously as number of attempts at intubation, use of additional airway devices or difficult laryngoscopy grade (cl iii, iv). it has been suggested that craniocervical extension relates to mouth opening and limited head and neck mobility can in poor mmp as mouth opening is limited and submaximal in neutral position. found that application of craniocervical extension improves the specificity and predictive value of mallampati airway evaluation. the sensitivity of two positions was 83% whereas addition of craniocervical junction increased the specificity from 70% to 80%. we found a sensitivity of 50 and 0% in normal and extended position in control group while the values for acromegalics were 13% in each position. the specificity 94% and 84% was seen in normal and extended position in control group while values were lower in acromegalic group (81% in neutral position and 65% in extended position). addition of neck extension did not improve the predictive value of mmp, which may be ascribed to the racial variation in the studied population, small sample size, or inter observer variability and criteria for difficult intubation. despite theoretical arguments that craniocervical extension relates to mouth opening and limited head and neck movement may restrict mouth opening, we were not able to find any additional benefit of neck extension while doing mmp for pre - operative assessment of airway in either control or acromegalic group. currently available bed side screening tests for difficult airway have poor to moderate diagnostic value when used alone.

: there are numerous reports of difficult laryngoscopy and intubation in patients with acromegaly. to date, no study has assessed the application of extended mallampati score (ems) for predicting difficult intubation in acromegalics. the primary aim of this study was to compare ems with modified mallampati classification (mmp) in predicting difficult laryngoscopy in acromegalic patients. we hypothesized that since ems has been reported to be more specific and better predictor than mmp, it may be superior to the mmp to predict difficult laryngoscopy in acromegalic patients.materials and methods: for this prospective cohort study with matched controls, acromegalic patients scheduled to undergo pituitary surgery over a period of 3 years (january 2008-december 2010) were enrolled. preoperative airway assessment was performed by experienced anesthesiologists and involved a mmp and the ems. under anesthesia, laryngoscopic view was assessed using cormack - lehane (cl) grading. mmp and cl grades of i and ii were defined easy and iii and iv as difficult. ems grade of i and ii were defined easy and iii as difficult. data were used to determine the sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of mmp and ems in predicting difficult laryngoscopy.:seventy eight patients participated in the study (39 patients in each group). both mmp and ems failed to detect difficult laryngoscopy in seven patients. only one laryngoscopy was predicted to be difficult by both tests which was in fact, difficult.:we found that addition of neck extension did not improve the predictive value of mmp.

the clinical, radiographic, and histological diagnosis of periapical lesions has been a challenge. current diagnostic methods help in fair assessment of accurate size and nature of a periapical lesion which determine the treatment and prognosis of the tooth in question. making the correct pulpal and periapical diagnosis is helpful for treatment and prognosis of the tooth. even if biopsy is the gold standard in differentiating granulomas from cysts, clinicians are aware of the difficulty in obtaining biopsies in routine clinical practice. therefore, there is an immediate need for a noninvasive method to diagnose lesions involving the periapical area. this could be due to fact that interpretation of images which influences success or failure also depends on several factors, including the clinician's experience in interpreting images. however, due to inherent drawbacks such as superimposition and distortion, it is sometimes difficult to detect periapical (pa) lesions on two - dimensional images. two - dimensional radiographs can image pa lesions to be detected only when bone mineral loss reaches 30%50% due to structured noise of superimposed cortices. therefore, only 5055% of small and medium sized lesions can be diagnosed as periapical disease. reports indicate that cbct images provide clinically relevant information not found in periapical images. a recent study used cbct as the reference imaging modality (gold standard) to compare the accuracy of periapical and panoramic radiography in detecting periapical lesions. the question that is valid to ask now is, does evaluation of a periapical lesion with cbct change the estimation of size and choice of treatment among endodontists compared to periapical radiography? selecting the most appropriate choice of treatment using the most accurate imaging modality would ultimately reduce cost and morbidity significantly in patients undergoing endodontic therapy. the purpose of this study was to compare lesion size and choice of treatment relative to the available radiographic information from periapical radiography and cbct. the null hypothesis is that there is no difference in the lesion size and choice of treatment of periapical lesions between conventional periapical radiographs and cbct images. the study group comprised twenty - four subjects, 11 women, and 13 men, with an average age of 53 years (range 1888 years) table 1. approximately one hundred subjects were examined to achieve a study sample of twenty - four. all subjects reported to the endodontic division of the university of detroit mercy school of dentistry with symptoms suggestive of a periapical lesion. history of present illness and clinical examination (including palpation, percussion, and cold test) of the tooth involved was performed before any radiographic procedure was undertaken. subjects were recruited to the study only after the initial intraoral periapical radiograph showed a periapical lesion of size greater than 3 mm. both single rooted and multirooted teeth with periapical lesion size equal to or greater than 3 mm on intraoral periapical radiography if a patient had more than one tooth that qualified for the study, the tooth with the most severe symptoms and maximum size of the periapical lesion as determined by conventional radiography was selected. previously root - treated teeth and teeth with restorations were also included in the study. endodontically involved teeth that had history of trauma or radiographic evidence of fracture were excluded from the study. no specific control group was used for either of the radiographic modalities tested since teeth other than the study tooth in the same jaw were also imaged and served as internal controls. the study was reviewed and approved by the university of detroit mercy institutional review board (udm irb protocol no . two - dimensional radiographs ( intraoral periapical) were obtained with an intraoral dental x - ray machine (planmeca intra, planmeca usa inc, il, usa) using the paralleling axis technique and round collimator (2.86 diameter) with variable kvp and mas and focal spot size 0.7 mm 0.7 mm. , long island city, ny, usa ) were used to record the images. schick cdr software was used to display images in real - time on a monitor. the images were stored as 8-bit tiff (tagged image file format) files at maximum quality (100%) (see figure 1). adequate image quality essential for diagnosis was established in a subjective manner after comparing previous images obtained from this radiographic modality and standardizedwith the ones that would be used for this study. the size of the periapical lesions was measured with a measurement tool available in the schick software. teeth with periapical lesions equal to or greater than 3 mm as seen on the two - dimensional radiographs were further recommended for a cbct scan. dimensional calibration for size was applied to the two - dimensional images upon export to the study software. calibration was applied using the calibration tool in the software in which all the images were observed. a particular distance was measured on the periapical image obtained upon exposure, and the same distance was measured when this image was exported to the software through which the images were available for the observers to view. after explaining the cbct scan procedure and obtaining approval from the patient for the imaging procedure, a cbct scan of the study patient was acquired using the 1719 platinum next generation i - cat cbct scanner (imaging sciences international, hatfield, pa, usa) and standard scanning protocols (pulsed exposure, 120 kvp, 3 to 7 ma, 14.7 second exposure time, 0.25 0.25 0.25 mm isotropic voxel size, 14 bit, maxilla or mandible) (see figure 2). collimation was fully adjusted to include the maxilla or mandible only. a limited (maxilla or mandible) fov (field of view) the fov for the maxilla and mandible was 16 cm (d) 6 cm (h). a cesium iodide flat panel detector was used to acquire images. only the arch (maxillary / mandibular) with the tooth and associated periapical lesion was scanned. all pertinent patient information on the scanned images was anonymized prior to being viewed by observers. scan data of subjects was exported from proprietary i - cat vision software (i - cat vision q, imaging sciences international, hatfield, pa, usa) to a previous version of i - cat software in.xstd format. images from both modalities were then imported into an interface viewing software, xv lite software (apertyx inc . ( figure 1 : intraoral periapical radiograph, figure 2 : cbct image as viewed by observers) the two - dimensional images were spatially calibrated according to known dimensions of the native images. periapical images were viewed in an office on a dell ultrasharp 2407wfp 24-inch widescreen flat panel lcd monitor (dell inc . , round rock, texas, usa), running under microsoft windows xp professional sp-1 (microsoft corp, redmond, wa, usa). the monitor specifications include a native resolution of 1920 1200 at 60 hz, pixel pitch of 0.27 mm, brightness of 450 cd / m, and contrast ratio of 1000: 1. the same monitor was not used for viewing both pa and cbct images for accession purposes. it was easier for observers to view cbct images at a monitor attached to the cbct scan acquisition hardware. the pa images were viewed by the observers at the principal investigator's office as these images could be easily exported from the endodontic department upon capture to that office. three endodontists were senior faculty in the department of endodontics and three of them were junior part - time faculty. the observers were asked to perform the following with the two imaging modalities separately: (a) measure the extent of the periapical lesion (greatest distance ( diameter) in millimeters and (b) choose treatment for particular tooth (root canal treatment, periapical surgery, root canal treatment and periapical surgery and no endodontic treatment). the observations were performed in two separate sessions: one for pa images and another for cbct images. this was done to enable observers could score all images between the two modalities in a timely fashion. all observers examined 24 images acquired from 24 different teeth (24 subjects) from the two imaging modalities. overall, a total of 288 scores were that is, 6 observers x obtained (24 images for pa + 24 images for cbct) each for lesion measurement and treatment choice. mann - whitney u test was used to assess for differences in the measurement of lesion sizes by all observers among the two modalities (p > 0.05). wilcoxon signed ranks test was used to look for differences in the measurement of lesion sizes among the two modalities by each observer. intraclass correlation coefficient was calculated among observers for each modality with respect to lesion size and choice of treatment. chi - square test was done to look for differences in the choice of treatment selected by all observers for the two modalities. the method of bland and altman was used to assess observer agreement of selected treatment between the two imaging modalities. the mean for lesion sizes among the two modalities for the study sample is shown in figure 3. the 95% confidence interval for either the lower or upper limit of agreement was less than two standard deviations (2sd = 22.86 = 5.72). table 2 shows no statistically significant difference between the two modalities in terms of the lesion size measured by individual observers (mann - whitney u = 9720.500, z = .916, p > 0.05). the intraclass correlation coefficient for the observers was 0.465 for treatment decision, and the high agreement (0.947) for lesion size. chi - square test revealed no significant difference in the treatment plan selected by observers between the two modalities ( =.036, p > 0.05 ). for some treatment decisions, there was equal or almost equal selection between the two modalities. figure 5 shows trend among observers in selecting treatments between the two modalities. the 95% confidence interval for either the lower or upper limit of agreement was less than two standard deviations (2sd = 22.86 = 5.72). this implies that the chance that observers chose different treatments from the two imaging modalities is minimal. the intraclass correlation among observers for each modality and both the imaging modalities together with reference to treatment selection and lesion size was calculated. (tables 4 and 5). though there was no statistical significance between the two modalities in terms of lesion size measured by individual observers , there was appreciable variability when the lesion size measurements of all the six observers were averaged (figure 6). with the introduction of cone beam ct in dentistry, several applications of this advanced imaging modality, including endodontics, are being investigated. in endodontics, the use of cbct involves diagnosis of pathosis, treatment planning, and diagnosis of trauma to dentoalveolar structures and evaluation of previously root - treated teeth, especially for missed canals. even though the benefits of cbct in endodontics are well understood, this particular study was undertaken to assess if the visualization of the third dimension as seen in cbct images would alter the choice of treatment made by endodontists. it is also interesting that the number of various treatment decisions (root canal treatment, periapical surgery, root canal treatment plus periapical surgery, and no endodontic treatment) was equal between the two imaging modalities. this led investigators of this study to believe that although cbct images show more information, this additional information would not necessarily translate into selection of a treatment different from the decisions made with the periapical images. in our study, six endodontists viewed images from both modalities (pa and cbct) and made their treatment decisions. the intraclass correlation with regards to the treatment decision was only moderate (0.465). this could be attributed to the varying clinical experience levels (novice to highly skilled). previous studies indicate that the long - term stability of observers in detecting periapical radiolucencies on conventional radiographs was satisfactory. two observers were well versed in interpreting cbct images and were using this technology on a regular basis while others had only knowledge of the cbct technology but were not well experienced in interpreting images. with reference to lesion size, there was, however, a high level of agreement (0.947) among observers between the two imaging techniques. this finding is in accordance with similar studies which confirm that periapical lesion measurements (when adequately calibrated) are the same between cbct images and traditional two - dimensional radiographic images. it is important to analyze the difference in radiation doses from the two imaging modalities under investigation in this study. several studies have assessed the effective radiation dose from different cbct scanners available in the market. comparisons in radiation dose between medical ct and cbct have also been made through scientific studies. the mean organ dose to organs such as the brain, thyroid, and parotid gland becomes extremely important while performing a cbct scan. cbct doses also differ with reference to the particular scanning protocol that is being selected for a specific study. short scan times and standard resolution (not high resolution) scan protocols would reduce the radiation doses from these procedures. the potential risk to the patient due to this increase in radiation dose can be minimized by using a limited field of view (fov). newer cbct scanners customized specially for endodontic purposes have the ability to scan only the particular tooth in question. however, in our study, the cbct scanner utilized did not have the ability to scan only the tooth and the entire jaw (maxilla / mandible) had to be imaged. the effective dose using the 2007 icrp (international council on radiation protection) calculation for the scanner and the protocols used in this study varies from 40 to 69 sv. the effective dose from the next generation cbct scanner used in this study is comparable to the dose from newer cbct machines currently being marketed only for endodontic purposes, for example kodak 9000, with an effective dose of 21 sv for a 4 5 cm jaw sextant. also, we used a scan protocol with an option to obtain higher resolution images since there was a need to display the best quality images for the observers to view. the scan resolution used in this study is similar to other studies of this nature. also lesion size differences between the two imaging modalities can be compared only when highly resolvable images are available. this study is not without its limitations: (i) the study sample (n = 24) is relatively small and it is not known if a larger sample would have produced a different outcome, (ii) history and clinical information (signs and symptoms) of study subjects were not provided to the observers at the time of the viewing sessions, so it is not certain if this information would have produced a significant difference in treatment selection between the two imaging modalities, (iii) the clinical experience of observers varied from novice to most skilled, (iv) the radiation dose from the cbct imaging procedure in the presence of an already existing radiographic procedure (periapical radiographs) should be validated, and a separate study comparing the effective dose of radiation used in cbct imaging and conventional radiography for endodontic purposes is required, and (v) it is not known if a different resolution of cbct images would have affected the since native resolution of cbct images was used by observers with no image enhancements (other than brightness and contrast) being performed for either modalities. it is now well recognized that prospects for the use of cbct in endodontics in the future appear promising. however, there are several factors such as diagnostic yield, radiation dose and selection criteria to be considered before this technology becomes incorporated into the diagnostic armamentarium in endodontics. since interpretation of cbct images requires additional training, care must be taken to avoid misinterpretation by clinicians who have not had the required training. for example, with an untrained eye, artifacts such as beam hardening due to metallic restorations on cbct images could be misdiagnosed as a carious lesion. endodontists aspiring to use this technology should either train themselves or seek the help of a maxillofacial radiologist who can interpret the images for them. also, when cbct images do not offer additional clues to diagnosis, periapical radiographs should be referred for more information. therefore, the notion that cbct would replace conventional periapical radiographs is far - fetched. another factor to be considered is the viability and cost - effectiveness of using cbct in a clinical setting. not all endodontic offices currently include cbct in their diagnostic work up. at the present time, the request for cbct scans for endodontic purposes is not uniform between specialists for the same reason. the cost of a cbct scan is several fold compared to a conventional periapical radiograph and the increase in cost needs to be justified. in , it was determined from our study that the choice of treatment in endodontics does not change significantly even with the inclusion of an advanced imaging modality such as cone beam ct. the of this study, however, should be interpreted with caution. although cbct performed almost the same as periapical radiography, the potential advantages of cbct in other areas of endodontics including assessing proximity of teeth to anatomic structures, such as mandibular canal and maxillary sinus, can not be underestimated. this study also proves the robust diagnostic potentials of traditional two - dimensional imaging modalities. further studies with a larger sample size are underway to further confirm the obtained from this study and identify appropriate areas in endodontics where cbct imaging would play a vital role.

objectives. to compare the ability of endodontists to determine the size of apical pathological lesions and select the most appropriate choice of treatment based on lesions' projected image characteristics using 2 d and 3 d images. study design. twenty - four subjects were selected. radiographic examination of symptomatic study teeth with an intraoral periapical radiograph revealed periapical lesions equal to or greater than 3 mm in the greatest diameter. cone - beam computed tomography (cbct) images were made of the involved teeth after the intraoral periapical radiograph confirmed the size of lesion to be equal to greater than 3 mm. six observers (endodontists) viewed both the periapical and cbct images. upon viewing each of the images from the two imaging modalities, observers measured lesion size and made decisions on treatment based on each radiograph. chi - square test was used to look for differences in the choice of treatment among observers. . no significant difference was noted in the treatment plan selected by observers using the two modalities (2 =.036, p > 0.05 ). . lesion size and choice of treatment of periapical lesions based on cbct radiographs do not change significantly from those made on the basis of 2 d radiographs.

in recent years, a variety of insulin regimens have been used for the treatment of type 1 diabetes mellitus (t1 dm) in children and adolescents. multiple daily injections (mdi) of rapid- (ra) and long - acting (l) insulin analogues have been used widely in these patients. moreover, use of continuous subcutaneous insulin infusion (csii) has been extended to those in whom mdi is either impractical or ineffective. these measures have been reported to improve hyperglycemia with reducing the occurrence of severe hypoglycemia in the young patients. on the other hand, the international society for pediatric and adolescent diabetes (ispad) recommends a target range for hba1c of less than 7.5% in pediatric patients of all ages with t1 dm in its clinical practice consensus guidelines. several studies have demonstrated that the recent hba1c levels of young patients with t1 dm on an intensive insulin treatment using newer insulins are still higher than the goal. in addition, it is known that optimization of metabolic outcome in females and adolescent patients is more difficult despite intensification of insulin therapy. we examined the annual mean hba1c levels during 2008 in japanese children and adolescents with t1 dm and compared them in relation to the sex and age of the patients and the insulin regimens employed to identify the factors associated with glycemic control. one hundred and three patients, 42 males and 61 females aged 16.8 7.0 yr, with a duration of diabetes of at least one year were enrolled in this study. the patients were divided into 5 age groups: group a, 14 yr (n=6); group b, 59 yr (n=17); group c, 1014 yr (n=18); group d, 1519 yr (n=26); and group e, 20 yr (n=36). they were also classified according to the insulin regimens used as follows: twice daily insulin injections, n=10; thrice daily insulin injections, n=14; four times daily insulin injections, n=49; five times daily insulin injections, n=15; and csii, n=15. in regard to the insulin preparations used, 8 patients used premixed insulin analogue (aspart+nph or lispro+nph), and 2 used twice - daily injections of a mixture of regular (r) and neutral protamine hagedorn (nph) insulins. six patients used varying combinations of premixed insulin analogue, ra (aspart or lispro) and l (detemir or glargine), 5 used premixed insulin analogue, ra or l, and one used thrice - daily injections of premixed insulin analogue and ra. in the 49 patients taking 4 injections daily, 42 (85.7%) used ra as bolus insulin and l as basal insulin, 3 used ra as bolus insulin and nph as basal insulin and 4 used ra and r as bolus insulin and l as basal insulin. in the 15 patients taking 5 injections daily, 12 (80.0%) used ra as bolus insulin and twice - daily injections of l as basal insulin, 2 used ra as bolus insulin and nph and l as basal insulin and one used ra and/or r as bolus insulin depending on their daily schedule and twice - daily injections of l as basal insulin. all the patients were instructed to self - monitor their blood glucose levels at home. determination of the insulin regimens and adjustments of the insulin dose were performed by one pediatrician, who was mainly involved in treatment of the patients at an outpatient clinic on the basis of the blood glucose profiles determined by self - monitoring and the lifestyles and daily activity schedules of the patients. the patients visited the outpatient clinic each month or at least every two or three months. we compared the annual mean hba1c levels from january to december 2008 in relation to the sex and age of the patients and the insulin regimens employed. the hba1c level was determined by an hplc method (normal reference range, 3.35.8%). the statistical significance of differences between groups was analyzed by the mann - whitney u test and kruskal - wallis test (dr . spssii). the statistical significance of differences between groups was analyzed by the mann - whitney u test and kruskal - wallis test (dr . spssii). the overall mean annual hba1c level in 2008 was 7.2 1.1% (4.59.7%). in the 103 patients, the mean hba1c level was < 7.5%, which is defined as optimal in the ispad clinical practice consensus guidelines, in 59.2% of the patients, while it was > 9.0%, which is considered to be predictor of poor outcome in the same guidelines, in 6.8% of the patients. there was no significant difference in the median hba1c level between the male and female patients (7.3 0.2% vs. 7.2 0.2%). in regard to the relation with age, the median hba1c level in group d was significantly higher than the levels in groups b and e (7.9 0.4% vs. 7.2 0.1% and 6.6 0.4%, p<0.05, respectively ; table 1). there were no significant differences in median hba1c level among groups a, b, c and e. in group d, hba1c levels of < 7.5% were achieved in only 8 (29.6%) patients; conversely, 6 (22.2%) patients showed poor glycemic control with hba1c levels of > 9.0%. table 1 differences in the hba1c level in relation to age at the time of the studyage groupnhba1c (%) group a (14 yr)67.6 0.2group b (59 yr)177.2 0.1group c (1014 yr)187.3 0.3group d (1519 yr)267.9 0.4*group e (20 yr)366.6 0.4*vs. the were expressed as medians se. * vs. groups b and e, the were expressed as medians se. in regard to the frequency of insulin regimens, of the 103 patients, 70 (72.8%) received three or more injections of insulin, and 15 (14.6%) received csii. among the 26 patients in group d, 22 (84.6%) used multiple injections of insulin or csii. in addition, all the young children in group a received either multiple injections or csii. table 2 shows the distribution of the median hba1c levels among the various insulin regimens. we could not find any statistically significant differences in relation to the insulin regimens employed. table 2 differences in the hba1c level in relation to the insulin regimens employedinsulin regimensnhba1c (%) twice - daily injections107.1 0.2thrice - daily injections147.6 0.3injections 4 times daily497.2 0.1injections 5 times daily157.0 0.2csii157.3 0.5there were no significant relationships between the hba1c values and the insulin regimens (kruskal - wallis test). the were expressed as medians se. there were no significant relationships between the hba1c values and the insulin regimens (kruskal - wallis test). there was no significant difference in the median hba1c level between the male and female patients (7.3 0.2% vs. 7.2 0.2%). in regard to the relation with age, the median hba1c level in group d was significantly higher than the levels in groups b and e (7.9 0.4% vs. 7.2 0.1% and 6.6 0.4%, p<0.05, respectively ; table 1). there were no significant differences in median hba1c level among groups a, b, c and e. in group d, hba1c levels of < 7.5% were achieved in only 8 (29.6%) patients; conversely, 6 (22.2%) patients showed poor glycemic control with hba1c levels of > 9.0%. table 1 differences in the hba1c level in relation to age at the time of the studyage groupnhba1c (%) group a (14 yr)67.6 0.2group b (59 yr)177.2 0.1group c (1014 yr)187.3 0.3group d (1519 yr)267.9 0.4*group e (20 yr)366.6 0.4*vs. * vs. groups b and e, p<0.05, respectively (kruskal - wallis test). in regard to the frequency of insulin regimens, of the 103 patients, 70 (72.8%) received three or more injections of insulin, and 15 (14.6%) received csii. among the 26 patients in group d, 22 (84.6%) used multiple injections of insulin or csii. in addition, all the young children in group a received either multiple injections or csii. table 2 shows the distribution of the median hba1c levels among the various insulin regimens. we could not find any statistically significant differences in relation to the insulin regimens employed. table 2 differences in the hba1c level in relation to the insulin regimens employedinsulin regimensnhba1c (%) twice - daily injections107.1 0.2thrice - daily injections147.6 0.3injections 4 times daily497.2 0.1injections 5 times daily157.0 0.2csii157.3 0.5there were no significant relationships between the hba1c values and the insulin regimens (kruskal - wallis test). the were expressed as medians se. there were no significant relationships between the hba1c values and the insulin regimens (kruskal - wallis test). good glycemic control is critical in patients with t1 dm. a target range of < 7.5% is recommended in the clinical practice consensus guidelines of the ispad for all pediatric age groups. the long - term microvascular and macrovascular complications of diabetes mellitus, the sequelae of acute hypoglycemia and the central nervous system alterations associated with hyper- and hypoglycemia are considered to be avoidable if this glycemic goal can be achieved. nevertheless, it still appears to be difficult to achieve this glycemic goal in young people with t1 dm, even when intensive insulin therapy using the newer insulins is adopted. danne et al. of the hvidoere study group on childhood diabetes reported that the mean hba1c level in 1998 of 2,780 children and adolescents with t1 dm aged 018 yr from 21 international pediatric diabetes centers in 17 countries was 8.62 0.03%, which was not different from that in 1995 of 2,101 patients aged 1118 yr from the same centers, despite adoption of intensive insulin regimens. de beaufort et al. of the same study group reported that the mean hba1c level in 2005 among 2,062 patients with t1 dm aged 1118 yr was 8.2 1.4%. they concluded that even intensive insulin regimens with increased use of csii were not effective for improving the hba1c levels in this large cohort of adolescents with t1 dm. these suggest that it might be difficult to obtain significant improvement of the glycemic outcome in young patients with t1 dm despite substantial changes in the therapeutic approaches. some studies have shown an association of poor glycemic control with the female gender and adolescent age . endocrinological factors play a possible role in aggravating glycemic control in females as compared to males, especially in the pubertal age group. in addition, females at this age suffer more frequently from behavioral problems, including eating disorders and subthreshold disorders, as compared with males. moreover, the practice of insulin omission to control body weight is frequently used by females of mid - teen age, which induces poor glycemic control. nonetheless, we did not find any significant differences in the median hba1c levels in relation to gender, even during adolescence, in the present study. on the other hand, we found the highest median hba1c levels among the patients in group e. most of the patients with an hba1c > 9.0% were adolescents. some studies have suggested that decreased peripheral insulin sensitivity during adolescence, perhaps caused by hypersecretion of growth hormone, is the major reason for deterioration of glycemic control at this age. moreover, the majority of adolescent patients with poor glycemic control had psychological and/or familiar problems in the present study. adolescence is a period in which young people try to gain independence from the family, but the demand for greater independence may not be equaled by the need to take greater responsibility for diabetes management. it may be difficult to motivate proper diabetes management during this period, but it is important for diabetes teams to elicit it by providing continual mental support and diabetes education (table 3). table 3 factors in deterioration of glycemic control during adolescence1)decreased peripheral insulin sensitivity, perhaps caused by hypersecretion of growth hormone2)corrupted lifestyles including irregular daily diets and activity patterns3)spread of psychological and/or familiar problems4)inequality between demand for greater independence and responsibility for diabetes management5)difficulties in motivation for diabetes management there was no significant relationship between the hba1c levels and the insulin regimens used in the present study; thus, the insulin regimen does not seem to have a major impact on the metabolic outcome in young people with t1 dm. nevertheless, we consider this to be one of the reasons why our patients were treated with optimal insulin regimens taking into consideration the patients lifestyles, customs and daily activity schedules, as well as the glucose profiles for the individual patients. patients who achieved satisfactory glycemic control even though they received twice - daily injections of insulin did not need either mdi or csii. for some reason, such as their lifestyles and daily activity schedules, they could not be treated with mdi; however, the twice - daily injections seemed to be more optimal for their treatment. patients who were treated with thrice - daily injections sometimes missed their bolus injections at lunchtime during school hours while on mdi with injections a times daily. this was the major reason why they chose the regimen of thrice - daily injections of insulin. on the other hand, patients on mdi or csii eventually required further intensification of the insulin regimens to improve glycemic control when the optimal glycemic outcome could not be accomplished by conventional insulin therapy. some patients who were treated with injections 5 times daily subsequently required twice - daily injections of basal insulin to attain stable glucose levels throughout the day. mdi and csii should be introduced in motivated patients of any age, including very young children, if they fail to achieve optimal glycemic control with twice- or thrice - daily injections of insulin (16, 17). consequently, the insulin regimen should be individualized for each patient, and an optimal insulin regimen should yield satisfactory metabolic outcomes. in addition, diabetic education and a team approach by the diabetes care team is indispensable to achieve good glycemic control. adolescents with t1 dm showed significant higher annual serum hba1c levels than patients from the other age groups. difficulty of diabetes management due to emotional issues and endocrinological factors during puberty may play a possible role in deterioration of diabetes control in this age group. on the other hand , the insulin regimen does not seem to have a major impact on the metabolic outcome in young people with t1 dm.

we examined the association between sex, age, insulin regimens and glycemic control in 133 japanese children and adolescents, 42 males and 61 females aged 16.8 7.0 yr, with type 1 diabetes mellitus (t1 dm). the patients were divided into 5 age groups and were also classified according to the insulin regimen. the annual median hba1c level in males (7.3 0.2%) was similar to that in females (7.2 0.2%). in regard to the age of the patients, the median hba1c levels in patients aged 1519 yr (7.9 0.4%) was significantly higher than those aged 59 yr (7.2 0.1%) and those aged 20 yr (6.6 0.4%, p<0.05, respectively). on the other hand, there were no significant relationships between the hba1c values and the insulin regimens. in , difficulty in management of diabetes due to emotional issues and endocrinological factors during adolescence may play a possible role in the deterioration of diabetes control. on the other hand, the insulin regimen does not seem to have a major impact on the metabolic outcome in young people with t1 dm.

mastery of the human reproductive process took another step forward in january 2013 when the american society of reproductive medicine (asrm) announced that oocyte cryopreservation was no longer experimental. several clinics in europe and the united states had reported births since 2000, and many programs were offering it without reliable evidence of efficacy. until january 2013 the asrm had called the practice experimental because the birth rate after thawing and insemination of frozen eggs was not as high as the birth rate from fresh or frozen embryos. professional recognition of the non - experimental status of oocyte cryopreservation is a noteworthy step in the history of assisted reproduction. with an already existing matrix of laws and practices for assisted reproductive technologies (art) the technology will also expand reproductive freedom in several important ways, particularly for women facing cancer treatment, wanting an egg donor, or seeking to preserve fertility against the ravages of time. those who argue that the field is under regulated will find a counterexample of sorts. while it empowers women in some respects, it creates unwanted pressure and alienation in others. those who gain the most may be the egg sellers and entrepreneurs who have emerged to fill and create the market demand for egg freezing. to illustrate these points , this article maps the developments in egg freezing, and shows how this evolving field is affected by and in turn may affect reproductive practices. as such, it may guide practice and policy for egg freezing and future reproductive innovations. part ii discusses the moral, medical, and social options that egg freezing offers to women. part iii examines how a market in frozen eggs might develop, though originally intended for personal use only. part iv looks at the effect of egg freezing on egg donation for infertility and research. part v comments on the problem of moving innovative therapies into a regulatory setting, usually through professional society guidelines. part vi concludes by showing how new reproductive technologies are shaping the meaning of reproduction even as existing attitudes and sex roles influence those uses. to understand the importance of egg freezing, one must first recall the problems with getting eggs, the difficulty in freezing and thawing them, and the variable success rates of clinics engaged in the practice. the role that the asrm has played in this story is deferred to the end. getting eggs, unlike sperm, has always posed special problem because of their relative scarcity and location in the body. although a woman is born with the maximum number of eggs that she will ever have, they are lost as she ages. the ability to hyperstimulate ovaries and then surgically retrieve multiple mature oocytes has been a driving force in the take - off of ivf and assisted reproduction. it has also made egg donation to infertile women and gestational surrogacy viable, as well as donation to researchers, and eventually for clinical use in regenerative medicine. sperm and embryos have long been frozen, often for lengthy periods, and then after thawing used successfully to initiate pregnancy. because of their larger size and volume of fluid, oocytes were not easily frozen and thawed. a slow freezing technique was tried first in the laboratory, and then with women who lacked other healthy embryos, but success was mixed compared to use of fresh or frozen - thawed embryos. crystals would form and interfere with meiotic spindle segregation in metaphase 2 of the oocyte development. a few art programs in italy (where legal constraints on discarding or freezing embryos gave incentives for investigation of egg freezing) eventually developed vitrification, a method of flash freezing to 270 f with a glycol cryoprotectant, which prevented the crystallization and meiotic disruption that had made slow freezing so unreliable. when the eggs were warmed and then a single sperm injected into cytoplasm (intracytoplasmic sperm injection ( icsi) ), higher success rates occurred. the technique soon spread to the united states and europe. with mounting data on live - births after vitrification , the asrm announced that egg freezing and thawing are no longer experimental in younger, highly selected populations for particular purposes. despite the asrm's pronouncement that oocyte cryopreservation is no longer experimental for several uses, programs vary in their skill and success rate in the art of freezing. the larger and more experienced programs will have success rates comparable to the use of fresh or frozen embryos, said to be 3050% per frozen - thawed egg, but few programs without considerable experience score that high. art programs that report offering oocyte cryopreservation, over 50% of them have never thawed and inseminated frozen eggs and had live births thereafter. as with other medical technologies, it is easy for practitioners to claim comparable expertise without showing comparable . the careful consumer will closely parse the numbers and pick only programs with well - established success rates. the asrm's pronouncement of non - experimental status for egg freezing empowers women in various ways, but it also risks selling them an ineffective service, particularly if they are in their mid - thirties or older, when their eggs will have already aged considerably. it may also distance them from the meaning of producing the female germ cells so necessary for reproduction by breaking the bond that exists between women as producers and consumers of their body's reproductive inputs, thus raising the commodification issues that weave though the ethics of assisted reproduction. in addition the asrm's approval of egg freezing empowers fertility programs and independent egg freezing firms with a new profit center and incentives to market that service. this section and the one following addresses these uses and comments on how egg freezing links empowerment and alienation in women producing eggs for freezing. egg freezing is a clear advantage for those couples undergoing ivf who have qualms about freezing embryos but few about freezing eggs. the qualms might be specifically moral, or they may reflect a more general concern about not having too many embryos left in the freezer. for either group egg freezing is empowering. they will be able to avoid the risk of reduced efficiency from an ivf cycle due to their reluctance to discard embryos or to have too many leftover. instead of limiting the number of eggs inseminated to those that they would transfer to the uterus (discarding the extra eggs or not retrieving them at all), now they may retrieve all eggs, inseminate only the number that can be safely transferred, and freeze surplus eggs for later thawing and use. if the stored eggs were no longer needed, they could be discarded without the ethical frisson of discarding frozen embryos. the catch in this approach is that there is no guarantee that the eggs chosen to be thawed and inseminated will yield the desired number for safe transfer and pregnancy, thus not providing the safety net they sought. the attraction to egg freezing is that if the first transfer is unsuccessful, they will not have to undergo another stimulation and retrieval cycle to obtain more eggs. there would be the additional cost in freezing and thawing eggs, but if a comparable success rate will be achieved, the price to the patient would be worth it. while empowering ivf patients with strong moral compunctions about embryo discard , egg freezing also opens the door to state disempowerment of patients with no such compunction. as right to life groups become more aware of art practices with fertilized eggs and embryos, which under their ethos are new persons who must be protected, they may support laws similar to those passed in italy that limit the number of embryos that can be created at one time and require that all embryos be transferred to the uterus. with extra eggs frozen and available for later thawing and insemination, such laws would limit the number of discarded embryos while, if the technique is effective, they would present no substantial interference with the efforts of an infertile couple to have a family. without the vatican in the vicinity, the political clout or inspiration to pass such disempowering laws in the united states may be absent. many people will view ivf as too far removed from abortion to spur legislation, particularly legislation that limits the ability of infertile couples to have families. nor would such limitations automatically follow from the state personhood amendments which periodically threaten but have not yet been passed. despite popular belief, merely defining the fertilized egg as a person under state law would not directly say what must be done with embryos outside of the body. to carry the discussion of constitutional issues a bit further, assume, however, that the state legislated directly to limit the number of eggs fertilized to the two or three that could be safely transferred to the uterus at one time, and required that all fertilized eggs be placed in the uterus. the purpose of such a law would be to reduce the chance that more embryos would be created and frozen, thus leading to their eventual death by discard or removal from storage. this justification would draw on the recognition in roe v. wade (and casey and gonzalez thereafter) that the state may protect prenatal life and potential personhood prior to viability as long as it did not substantially burden the core right to abortion. since the embryos are not in the body, embryo protection laws would not fall under the abortion right recognized in roe. but such laws could limit the safety, efficacy, and cost of ivf to such an extent that it interfered with a woman's right to use ivf to have a family. while not yet recognized as such by the supreme court , many scholars would argue that that right is founded in a more general privacy or liberty right to have a family, and not a right to terminate a pregnancy as such. on this view a law limiting the number of eggs fertilized, requiring that all be transferred to the uterus, and prohibiting freezing embryos would appear to be a substantial burden on that right. yet if egg freezing is no longer experimental, the state could argue that eggs not fertilized could be frozen and inseminated at a later time, without having to go through another hyperstimulation and retrieval cycle does not burden or infringe the right at all. as we will see in part iv, the non - experimental label is a misnomer of sorts. yes, some programs have very good success rates after thawing and insemination, while others have had limited or no experience with egg freezing even though they offer it. favorable success rates have usually occurred with younger, healthier women and not older women or those whose fertility problems are related to egg quality. given the uncertainties of whether the two or three eggs inseminated will cleave and reach the stage appropriate for transfer, limiting the number of eggs inseminated with no option to inseminate more and freeze the extra embryos might mean that a woman ends up without enough viable embryos from that cycle to achieve pregnancy. she would then have to go through another costly and burdensome stimulation and retrieval cycle. until egg freezing were a comparable effective substitute for embryo freezing, such a policy would arguably so burden the woman's freedom to reproduce as to be unconstitutional. this argument assumes that close or substantial comparability of efficacy would be required for a law limiting the number of eggs inseminated to be constitutional. recent circuit court cases on abortion restrictions, such as the constitutionality of hospital privileges requirements and following the fda label for medical abortions, indicate otherwise. the 5th circuit in a recent texas case refused to uphold the preliminary injunction issued by the district court simply because the law at issue made abortion more expensive or harder to access, on them ground that the situations before and after the new restrictive law. if the supreme court applies this more lenient standard to abortion laws, it is likely to do so for laws that interfere with infertile couples having children by restricting the number of eggs inseminated or embryos transferred. egg freezing also enhances freedom for women undergoing cancer treatment who want to preserve their fertility. if they had a spouse or partner, they could undergo a stimulation and retrieval cycle, create embryos, and freeze them for later use. they could do the same without an available partner, if they were willing to use donor sperm to create embryos. if they lack a partner and willingness to use a sperm donor, egg freezing would be empowering because it both protects their fertility and gives them choice over the genetic father of their post - treatment children. a similar need might arise with women with genetic diseases or other conditions, such as premature ovarian failure, who had not yet found a partner but wanted to ensure they had healthy eggs at a later point in their life for reproduction. offering egg freezing to cancer patients involves the disadvantage of needing a controlled stimulation cycle to harvest eggs, which delays chemotherapy and may lead to high patient estrogen levels. while exposure to more estrogen is generally not a good idea for estrogen sensitive breast cancers, the amount of exposure during one retrieval cycle might not be so great as to counterindicate it. with the time interval between diagnosis and the start of treatment varying among malignancies the controversy among clinicians, especially oncologists, about the risk of high estrogen levels might lead some clinicians and patients to attempt single egg retrieval in a natural cycle or immature oocyte retrieval with in vitro maturation of early stage oocytes. information is sparse about whether women who have used this option have been able to successfully preserve their fertility, mainly because so few have attempted pregnancy with thawed eggs after treatment. but a few babies have been born, and as long as treatment is not unduly delayed and ovarian stimulation is not contraindicated, few would argue against this option. the risk that it might lead to children whose mother dies from a recurrence of the cancer is not a sufficient ground not to proceed. egg freezing has also been touted as a way to provide women who are still fertile with insurance against their biological clock. women in their twenties or early thirties will be able to devote themselves to career without losing reproductive potency. by freezing eggs they will still have healthy eggs available for when they are ready to start a family. rescheduling motherhood in this way does sound empowering, but again nothing is as simple as it sounds. imogen goold and julian savelescu and others have articulated the general case for non - medical or social egg freezing. egg insurance against future infertility will enable equal participation in employment, allow women time to find a compatible partner, and enable them to postpone pregnancy until they are emotionally and psychologically ready to have a child. assuming safety and efficacy their arguments are generally persuasive but need to be broken down into the differing perspectives that different age groups of women will bring to egg freezing. the equal participation in employment argument emphasizes the importance of enabling women to obtain an education and invest in career without losing the opportunity to have children. a more desirable approach to this dilemma, of course, would be to change traditional employment models so that women do not face restrictions from time off for child - bearing and rearing. egg freezing is no substitute for those efforts, but it may provide some women with reassurance that they can commit themselves to education and work without losing their fertility. with marriage and children such a dominant cultural narrative for women, they may be more concerned about their relational status, their ability to find a suitable mate, and whether they are emotionally and psychologically ready for children. this pressure weighs even heavier if they are at risk for premature ovarian failure or other medical conditions that will limit their supply of healthy eggs. age group differences will arise with both careerist and relational concerns. to ensure the greatest fertility , it would be best for women to freeze their eggs in their early or late 20s. at this point many women but not all would usually have completed their education and started on a career. if they have not yet had children, and have no partner or immediate plans to do so, banking eggs would appear to enable them to live their lives without the biologic clock ticking so loudly in their ears. also, since health insurance is not likely to cover egg freezing until its efficacy is much better established, some of them might have the reources to cover the costs of thawing, insemination, and caring for a child. yet the optimism bias of the relatively young may make the risk of future infertility seem quite distant. surely, they say to themselves, they will find a man and settle down in the next few years, so why undergo the intrusion and cost of egg freezing? only the most risk averse or those with a yen for the latest technological fix, may be willing to take the hormones and pay out cash for the egg insurance that they may never need to cash in. the early twenties perspective on women in this cohort may become acutely aware of the loss that they will experience if they postpone conception too long. if they are not in a relationship conducive to having children, a cold look at the facts may lead them to stock the egg freezer. as they slip into their late 30s, the internal pressure to freeze eggs will grow. at this point egg freezing may still their anxiety and allow them to get their workplace, relational, or psychological states in order. yet freezing post-35 may not give them the fertility they hope for, if only because the viability of their eggs will also have diminished. preferences here will vary with the importance of having children with their own eggs versus that of a donor and the varying degrees of reliability of actually producing children with different techniques. egg freezing for teenagers and those in their 20s is the most likely to be successful but the least likely to be needed (stimulation and retrieval, not to mention cost, is hardly trivial). for women in their late 20s or early 30s one alternative would be to do a retrieval cycle, freeze some of their eggs, and inseminate some with donor sperm and then freeze those embryos. when they are ready to reproduce with a partner, they can thaw the frozen eggs and try with those. if that does n't work and donor sperm is acceptable to them, they can use the previously frozen donor sperm inseminated embryos. or they could use a donor egg and their partner sperm, and if no partner, an egg and sperm donor (or already created embryo). the same would go for women who freeze after 35freeze some eggs and create embryos with donor or partner sperm, and then when ready for children, use the frozen eggs and then frozen embryos. if none of those work, then they still have the option of using donor eggs with partner or donor sperm (or buying an already created frozen embryo). sarah elizabeth richards, a strong proponent of freezing eggs, ignores in her fervency the lower success rate with freezing eggs after 35 or these other options for motherhood. her 2013 book motherhood rescheduled: the new frontier of egg freezing and other writings present egg freezing as a culture narrative shift that allows women to one is struck, however, by how she oversells the technology as relief from the angst of finding the right mate and having children. richards begins one op ed (why i froze my eggs ( and you should, too) ) recounting how between the ages of 3638 she spent $ 50,000 of her savings to freeze 70 eggs in the hope that they would help her have a family in her mid-40s when she would be ready with a mate. for richards it was the best investment she ever made, because it stopped the deep sadness that without a reliable partner she was losing her dream of being able to have children. egg freezing snapped away the punishing pressure to seek a new mate before she was ready. the most powerful gender equalizer of all the ability to control when to have children. just as men in their 40s (or even later) could have children, she could too. here's the rub. $ 50,000 for egg freezing at 37 is an expensive and probably ineffective way to quiet the ticking fertility clock. if a woman is listening carefully, this move will only lessen the thrum, not quiet it altogether because the viability of late-30s eggs is much less than that of eggs frozen in one's 20s or even early 30s. none should think this form of insurance will cash out with the child that they want. with or without egg freezing they will be able to bear and rear children, though perhaps not with the genetic tie that egg freezing provides. the rescheduling motherhood narrative, so empowering for some, might be disempowering for others. rather than you may choose to freeze, some might read the message sent as this aspect leapt to the fore when a report gurgled up from the internet about a top law firm that offered egg freezing to new associates. some saw here exploitive law firms wanting to feast on the energy of lower paid lawyers by reassuring them that devotion to the firm would not wreck their family plans. no worries, they could simply put them off with egg freezing. whatever this hypothetical firm's agenda , it will be important that egg freezing be structured as an opportunity not a requirement (though the two may be difficult to separate). young women wanting to start families earlier rather than later should be protected in that choice, and not pressured to think they must defer children because of the demands of launching a career. indeed, if their employer or medical insurance is paying, it may create even more pressure to do so, even if the firm is simply trying to stay competitive. and entrepreneurial art programs or egg banks will market this service to the relevant age groups. expect broadcast, print, or internet advertisements that play on infertility fears and offer freezer discounts. egg freezing and other reproductive technologies are only empowering if they allow women (and men) the choice to use them. egg freezing fails that test if it is read as a prescription that all women entering the workforce or reaching a certain age should store their eggs. indeed, the chance to use it will also require the $ 10,000 to go through a stimulation and retrieval cycle to freeze eggs and pay ongoing egg storage costs as well as a tolerance for the medical intrusion involved. and a perception that egg or embryo donation, which lacks the genetic connection of using one's own eggs, will not fill the gap. as more women freeze eggs for social reasons, the question of what they will do with their eggs when no longer wanted will arise. will they discard them? donate or sell them to other women, to egg banks, or to researchers? similar dispositional questions will arise with eggs frozen by women not wanting to create more embryos than can be transferred, or by women facing cancer or other health issues. here egg freezing empowers women to assert ownership and control over the eggs they have produced. women banking eggs are the owners of them, and will have ultimate dispositional control over them until they choose to transfer that control to another. how they will or should exercise that control is the question. egg freezing will thus be a legal as much as a medical or psychosocial transaction. the women involved will sign a contract with the bank about their rights to remove their eggs at any time and how they should be disposed of in the case of death, failure to pay storage fees, or other contingencies. presumably they may write a check on their egg account, designating themselves or another as payee. egg banking thus raises the possibility that egg storers will sell their eggs to other women or researchers who are in the market for egg donors and find those eggs cheaper or desirable for other reasons. the national organ transplantation act allows buying and selling eggs and sperm, though a few states have a ban. unlike eggs stored at a later age when women are going through infertility or stored for cancer treatment, eggs stored by women in their 20s or earlier 30s may be as good as healthy donor eggs and now available at a better price. if they are of an entrepreneurial bent or simply want to recover some of the costs they have invested in egg freezing, one can expect some women who freeze their eggs to participate in the market for donor eggs. of course, with so many eggs available from storage, the price of eggs may drop or rise depending on the fertility, health, and genetic desirability of the woman offering the eggs. laboratory and biobank regulations to ensure health and safety of storage facilities beyond existing fda regulations are likely to follow, as well as liquidity and other banking controls. as women come to view storing eggs as a banking transaction, with the possibility of accruing interest (in the form of rising prices) or independent sale, a sense of alienation or commodification might accompany egg freezing. although their goal at time of freezing is to reserve their own fertility, they will also have to think of their eggs as possible future commodities that are transferred to others, either on grounds of altruism or profit. the forms they will be asked to sign for future disposition of stored eggs will remind them of this contingency as well as the infectious disease and other fda screening that will then have to be done to prepare for or done at a later time for those contingencies. at some point women freezing their eggs may come to see them as another commodity on the reproductive market / that arose from their non - market efforts to preserve their fertility. only later are do they become marketeers, either as entrepreneurs or sellers in their own right, but that possibility hovers from the inception. freezing eggs solely for infertility purposes and then selling them later may be less alienating than if sale is intended from the start (eg going through a retrieval cycle for the purpose of selling eggs). a greater degree of alienation may also enter the already partially alienated practice of egg donation. as the next section shows, the gift frame that largely surrounds paid egg donation for both donors and recipients will shift toward a more commercial frame, akin to the frame that now surrounds sperm donation. this is not harmful in itself, but frame shifting will necessarily require adjustment in the social and psychological valence that eggs now carry. the work she has undergone is no longer for herself alone, once she decides to sell them. as eggs become a commodity on the market, gender may be less important than it previously was. fungibility will also be a challenge. because of the genetic tie and its meaning for women banking their eggs, egg are not fungible. they will also vary in other genetic endowments, eg, mutational load for common and rarer disease and the age and viability of the eggs. except for the family tie , eggs from women of comparable ages and health may be fungible but of little interest to others because they are older and not that much less expensive than egg donors recruited for that purpose. egg freezing empowers women by giving them a way to preserve oocytes against the threat of age or disease. it also empowers the doctors and firms that provide those services, opening additional ways to sell art services and egg banking itself. a particular aspect of that empowerment is the creation of a novel kind of biobank, with a set of issues different from those that arise in tissue, dna, and sperm banks. to store eggs to use those eggs for reproduction, she will need a specialist in thawing and inseminating those eggs, and then placing them in her uterus. egg freezing will thus increase demand for art services, some of which would not otherwise have been requested. the specialists providing art may add egg banking to their portfolio of services, contract it out, or refer to established banks. since eggs will need to be frozen after retrieval, the art programs are likely to do that themselves. they will also prefer to thaw, inseminate, and implant embryos because of the additional charges generated. actual storage of the eggs might be at the clinic site or contracted or referred off - premises, as increasingly occurs with frozen embryos. as egg freezing proliferates, independent banks might emerge to handle administration of deposits, storage fees, and sending frozen eggs per depositor instruction to a site or program where thawing and insemination or other disposition will occur. regulations to ensure proper storage, accounting, and response to depositor instructions may be needed. legal protection for loss of stored eggs should also be provided, perhaps based on the cost of production. in the early stages of egg freezing there will be few depositors and few non - clinic egg banks. as the practice grows and art clinics get out of the longterm storage business, the number of off - premise egg banks will grow. the use of frozen eggs from paid egg donors will also give a boost to expansion of this sector. at some point full service reproductive banks might develop, with one entity banking eggs, sperm, and embryos, or eggs and embryos. when thawing and use is desired, stored gametes or embryos may then be transferred to the art programs that specialize in thawing, insemination, and implantation. reproductive biobanks serve different purposes than the many biobanks that now exist for tissue, dna, and other samples, but common issues arise. all biobanks have to face issues of who may deposit, the conditions of deposit, who may withdraw samples, legal protection for the privacy and quality of the sample, and how rights or claims in products that arise from the banking (less important for reproductive banks) are allocated. egg banking focuses attention on the choice of the depositor because she must invest money and body to obtain the banked entity. her physical, monetary, and emotional investment in the deposit may be greater than someone giving a sample of dna or tissue from an operation for research. her ability to have her genetic child may ride on the deposit, which itself may have an alienating aspect from her embodiment that other biobanking does not. other banks may focus more on research into prevention or treatment of health and disease, potentially affecting thousands, if not millions of persons. dna biobanks to uncover gene associations with common or rare diseases, such as the 1000 genome project in the united states or the uk biobank, have great research significance. other biobanks may amass tissue that could be used in treatment, such as the embryonic stem cell bank proposed for racial and ethnic variety. some banks may be more concerned than are gamete or embryo banks about intellectual property rights in ing discoveries or the need to contact depositors about later annotations or calls on variations of unknown significance. each instance of banking will have its own set of challenges that depend on the precise use at issue. only reproductive banks will enable an infertile woman or couple to have a child, and need to be regulated accordingly. egg freezing will also empower the participants in the current system of paid egg donation, though less the egg donors themselves than the entrepreneurs and commercial providers. these effects, however, may make finding egg donors easier or more efficient, thus empowering recipients who lack eggs to have families. an additional benefit is that it will increase the supply of donor eggs sufficiently so that professional guidelines calling for informing or not using egg donors who have been through six donation cycles might not so drive up the price of eggs that they become out of reach for some women or lead to harmful effects on repeat donors. the asrm's 2013 statement cited better coordination between egg donor and recipient as a reason for finding egg freezing non - experimental. no longer would it be necessary to have the donor and recipient's cycle synchronized so that retrieval, fertilization, and embryo transfer take place at the same time and place. now the donor eggs could be retrieved and then frozen, and thawed and inseminated at the most convenient time and place for the recipient without fretting about donor compliance for synchrony with the recipient's uterus. egg freezing, however, may facilitate more far - reaching changes in the egg donation system. that system increasingly relies on brokers to identify donors, match them up with recipients, and then have the stimulation, retrieval, insemination, and transfer done by the recipient's physician. with the increasing reliability of egg freezing, especially from younger women, egg bank entrepreneurs may recruit several donors of differing ethnicity for egg production, have their eggs retrieved, genetically screened, and then freeze them for later sale as demand for eggs of women with that donor profile arise. the egg bank (which might be run by a fertility doctor with experience in egg freezing and egg donation) will pay the donor's fee and retrieval costs up front for an initial supply of eggs. since one donor could produce 1025 eggs, it would be possible to sell frozen eggs in batches of 46 to women seeking them at a cheaper price than if recipients had hired the egg donor and her entire output themselves. the thawing and insemination could occur at the frozen egg bank (and its associated doctors) or could be shipped to affiliated programs which are trained in the bank's thawing techniques. the upfront investment in egg production might lead to greater number of sales of eggs per donor, thus increasing the egg entrepreneur's profits. an atlanta ivf program had tried for many years to develop safe and effective egg freezing. the program owners then decided to start a commercial egg bank that would recruit egg donors and sell batches of six eggs to couples / women for half the price of a recruited egg donor. its costs of recruitment, retrieval, and donor fees, which would roughly be the cost for a donation to a single donor, could be recovered by selling the now frozen donated eggs to several donors. depending on the number of eggs retrieved and the demand for a donor with those characteristics (age, education, looks, proven fertility, etc) a single donation via the frozen egg bank route could make egg donation cheaper for recipients and more profitable for the egg agency / art program. it would also lead to fewer women acting as donors, since one donor could provide eggs to several recipients. such a change in the egg donation system would entail some reframing of how egg donation is currently perceived. as professor rene almeling shows in her insightful study of egg and sperm donation, egg donation is now framed as a gift to infertile women, albeit for a fee. while the money is the key to women choosing to be egg donors, the gift nature of the situation is also important, and very different than how sperm donation is framed by sperm banks and experienced by sperm donors. would donors be as easily recruited for contributions to a frozen egg bank, which then sells their eggs in batches to recipients? although this change would inject more commodification into egg exchanges, the donors would still be helping infertile women, indeed, more than the one that their donation would traditionally have aided. their need to meet or know about the recipient could also be met by providing that information prior to the donation and informed of it when other women wish to buy their eggs from the bank. key here, of course, is whether this system would maintain or increase demand for frozen relative to fresh donor eggs. would a woman or couple seeking an egg donation be willing to forego the notion that the donor donated just for them? if the success of egg freezing is comparable to initiating successful pregnancies, information is available about the donor, and the price is lower, the use of donor eggs from egg banks might well meet the needs of recipients, and thus reinforce this practice. it may also make it easier to keep track of half - siblings born as a and enable contact with the donor, if she has chosen identity release on the eighteenth birthday of offspring. indeed, it may lead to a much greater use of donor eggs obtained from egg banks, which will cost less and may give a greater range of choice, though it will lack the chance to meet or talk with the donor, as now sometimes occurs. a further alienation from the body and the person may . the expansion of egg donation in this way depends on the legal infrastructure for ownership of eggs. it assumes that the egg donor is transferring all of her ownership interests in her eggs to the egg donor entrepreneur who recruits her and pays her fee and the costs of stimulation and retrieval, and then sells or transfers her eggs to others as he or she finds fit. the donor would retain no rights, nor would the recipient who previously would have gained title to all of them, since the recipient would be purchasing only a portion of the donor's output. there is a gain in efficiency here at a cost in greater alienation or commodification of gametes, though it may also be easier for ing offspring to contact each other and their genetic mother. in the transition period before such arrangements proliferate, couples or women who arrange for an egg donor will ordinarily be paying for and acquiring her entire output. but they may not need all of them, and instead of inseminating all, might choose to share (for a part of the expenses) with other women looking for an egg donor. this might be feasible in a fresh cycle for both, but logistics might be easiest if the recipient freezes eggs that she does not use to create embryos. as purchaser of the donor's output in that cycle, she would be free to freeze some of them and sell or share with others. this would turn the recipient into an entrepreneur who then sells some of the eggs she purchased from the donor to another. to facilitate such a market, egg banks specializing in donor eggs may buy her surplus and sell accordingly. no doubt other problems will arise as the field grows, beyond the issues already noted. a shortage of eggs has plagued some embryonic stem cell researchers, in part because of bans on paying egg donors (even though egg donors for infertility can be paid). the successful production of embryonic stem cell lines through somatic cell nuclear transfer will spur demand for research eggs as will the uk's recent approval of experimental treatment of mitochondrial dna defects using defect - free partial egg donors. with a growing number of women freezing and banking their eggs, eventually some of them will donate unwanted eggs to researchers, to banks that will provide them to researchers, or paid egg donors will be recruited specifically to donate to research, with unused eggs at the time frozen for later research. a main barrier to the shortage of research eggs has been ethical concerns about the propriety of paying egg donors for research eggs, even though it is now well - accepted that paying women who donate eggs to infertile women is acceptable. the legal prohibition on paying for organs never extended to blood and gametes, and only a few states ban payment for eggs. in the united states, however, the controversy over federal funding of embryonic stem cell research led to a funding policy of not funding research with cell lines derived from embryos created from paid gamete donors. several states followed suit by banning any use of paid gametes in stem cell research, even though they allowed fertility donors to be paid. new york has now removed that ban but governer jerry brown vetoed such a bill in california. in any case, the federal limit on paying gamete or embryo donors in federally funded stem cell research still remains. with a growing number of women freezing and banking their eggs, eventually some of them will donate unwanted eggs to researchers, to banks that will provide them to researchers, or paid egg donors will be recruited specifically to donate to research, with unused eggs at the time frozen for later research. freezing technology will not dissolve moral concerns about paying egg and sperm donors, but it should make eggs so widely available at a reasonable price that many researchers will be able to go forward with non - federally funded research. as scientific interest in using oocytes in research takes off, the availability of eggs from women who have banked eggs initially for themselves and from donors recruited for that purpose should meet most scientific needs. the movement of egg freezing from research to innovative therapy to accepted practice illustrates the trajectory of much medical development and the pitfalls along the way. the united states does not have a national fertility regulatory agency to announce when success rates with egg freezing are acceptable, nor are such judgments within the jurisdiction of the fda. as a , it fell to a professional society of doctors offering ivf (and egg freezing) to play that role. indeed , it took that responsibility quite seriously because of earlier criticism from a presidential bioethics council that too many new reproductive technologies were being used without adequate testing. basic ivf itself was highly innovative and full of risks to ing children, but when it succeeded in 1978, doctors inside and outside the u.k. the ability to hyperstimulate the ovaries and remove several eggs, fertilizing and transferring some embryos to the uterus, and freezing surplus embryos for later use, gave a great boost to the field. the use of icsiwas also introduced without controlled trials, first for problems of male infertility, and then for a wide array of other situations, so that it is now used in 50% of basic ivf. many other aspects of art have also been adopted without controlled trials or systematic investigation, including different embryo cultures, the use of assisted hatching, when to transfer embryos to the uterus, preimplantation genetic diagnosis, egg freezing, and the like. art is not exceptional here this pattern is typical of many areas of medicine. to deal with criticisms that too many assisted reproductive innovations were being used without adequate study the asrm issued a report in 2009 on the definition of experimental procedures. it proclaimed that procedures, tests, treatments or other interventions for the diagnosis or treatment of infertility are considered experimental or investigational until the published medical evidence regarding their risks, benefits, and overall safety and efficacy is sufficient to regard them as established medical practice. medical evidence relevant to determining whether something is established medical practice must be derived from appropriately designed, peer - reviewed, published studies performed by multiple independent investigators. i d. it went on to say that the asrm will state specifically in official publications that a procedure is considered experimental or investigational and will remove a procedure from that status when evidence warrants it. the asrm also warned that procedures classified as experimental or investigational should not be represented or marketed to patients as established or routine medical practice. advertisements and other materials describing or relating to that procedure should state specifically that the procedure is not established medical practice and is classified by the asrm as experimental or investigational. patients choosing that procedure should be counseled about the experimental nature of the procedure and have that counseling documented. the asrm report, however, does not require that a physician offering an investigational procedure do so only in the context of a research study. an experimental treatment must be labeled as such, regardless of whether it is offered as part of a research project blessed by an irb. the asrm's handling of the problem of when an experimental or innovative practice moves to established status illustrates some of the issues that arise with use of a professional association to make those decisions. issuance by a self - interested and resource - limited professional association risks establishing minimum rather than optimal standards of care, with no effective means of enforcement. despite best efforts, those standards may be internally inconsistent, are time - dependent, and may be quickly outmoded. unsurprisingly, they may also reflect the interests of those issuing the guidelines or their sponsors. several of these problems are evident in the asrm guideline on egg freezing, a practice initially offered or marketed to women without good evidence that it was safe and effective. as noted, the asrm practice committee had taken the position that egg freezing is experimental and not ready for prime time use because the live birth rate was significantly less than the success with fresh or frozen - thawed embryos. when it announced in 2013 that egg freezing was no longer experimental in younger, highly selected populations for particular purposes, it based its both on preliminary data on safety and four random controlled trials of fresh versus vitrified / warmed oocytes, finding that implantation and clinical pregnancy rates are similar in young, highly selected populations to success rates with fresh eggs or frozen - thawed embryos. it noted, however, that these may not be generalizable, and clinic specific success rates should be used to counsel patients whenever possible. also, large observational studies of clinical practice suggest that success rates overall may be lower. while finding that egg freezing was no longer experimental for patients receiving gonadotoxic therapies for cancer or other diseases, by woman with genetic disease, and couples with ethical or legal constraints on freezing embryos, it took a different position with regard to egg freezing to circumvent reproductive aging in healthy women. it found no data to support the safety, efficacy, ethics, emotional risks, and cost - effectiveness of oocyte crypreservation for this indication. one may question why the same data that supports recognition for approved uses would not also support recognition for this use, other than the population of older women who been the main customers for egg freezing. so how to explain the asrm's inconsistency in 2013 to certify some uses of egg freezing as non - experimental and others, such as social uses of egg freezing, as still experimental. in its view there was no data to support the safety, efficacy, ethics, emotional risks, and cost - effectiveness of oocyte crypreservation for the sole purpose of circumventing reproductive aging in healthy women. presumably the asrm would like to see studies of women who have banked eggs for this purpose, especially over age 35, who were its main customers so far in the the data was not overwhelming for the non - experimental uses that had been approved, simply because there had been so few studies. the data for social uses (the health effects of doing so, the live - birth rate of pregnancy after freezing and thawing, psychosocial reactions, and more) will not be immediately forthcoming because those uses have been relatively few, offspring born even fewer, and studies of them non - existent. initially the asrm's hedge on freezing as social insurance smacked some women as less consumer protection than old - fashioned, male - dominated medical paternalism. women were quick to argue that they should be informed of that option so that they could make their own choice. in fact, behind its labeling rhetoric this was all that the asrm was urging: fully informed choice. the asrm feared that some clinics or agencies would tout social uses as a way to sell ivf cycles and egg banking services, when there are too many uncertainties to justify the costs and physical burdens of doing so, particularly for women over 35 who appeared at this time to be the most likely users. some uk doctors agree with this position because of lack of benefit to justify the harm imposed. in addition, if programs were not fully informing patients of egg freezing's non - established status, they would be violating the principle of informed consent. the asrm's position was actually less protective than it purported to be, which is one of the problems with professional self - regulation. art programs could not sell egg freezing as standard, established therapy, but they could advertise and sell it as long as they labeled it truthfully as experimental. nor did it require that a program bring egg freezing to an irb as a research study. in the end, informed consent would be the watch word here, as seemingly everywhere else in bioethics. a similar strategy is seen here in the asrm's practice committee's discussion of limits on the number of donor egg cycles. in the abstract , the practice committee does not say that more than six cycles should not be done, but only that donors be informed of the possibility of undocumented but possible health risks. in the it states that it is prudent to limit the number of stimulated cycles to approximately six. the slushy language here shows another limitation in professional society regulation, in this case to try to avoid mandatory statements that are based on insufficient data or that could lead to charges of antitrust collusion. the map provided here of how egg freezing fits into the ethical, legal, and medical matrix of existing art practice has mentioned several kinds of regulation that might apply or be needed. there already are fda guidelines on handling donor gametes in the laboratory, which will apply if women who freeze eggs for their own use wish to sell or donate them to others. if egg producers had not been tested for various infectious diseases at the time of freezing, they will have to be tested before their eggs can be used by others. existing state laws such as those in california and in new york will be a model for ensuring safe operation, as will the federal clinical laboratory improvement act. but egg banking raises special issues concerning the depositor's need to be sure that deposited eggs are safely preserved and returned to the depositor or to a designated recipient. there will be many similarities and differences from traditional cash banking, which may yield some insights about how egg freezing and other forms of biobanking should be addressed. an issue of general regulatory concern with biobanking is what damages if one's banked sperm, embryos, cord blood, or other tissue has been lost, stolen, or damaged. because of the family importance of the banked materials or their need in specific medical procedures, money damages may be hard to calculate and will have an alienating effect on those persons who have lost banked material. beyond health, safety, and banking issues, there is a need for clear recognition of the woman's ownership of and hence dispositional control of her eggs. while such a would seem to follow from previous cases and practices, any regulatory regime should start with recognition of her legal rights in her eggs. until she has validly transferred full or partial control to a spouse, partner, physician, bank, or researcher, it is her right to decide whether to produce eggs, store them, have them inseminated, or transfer full or partial control to another. by the same token , once she does so, she loses that control and may have no further say in whether they are used to produce research embryos or embryos for infertile persons. a full regulatory regime is lacking for many aspects of art and egg freezing is no exception. women, however, play a special role both as providers of gametes and as gestators. no man can reproduce without an egg source and/or gestator, while women can gestate without using their own egg. traditionally, they have also played a much larger role in rearing, though more involvement by fathers is occurring in the united states and in many european countries. art expands both women's career, education, and fertility options, and makes special demands of them. sometimes they will resort to art to help the man overcome his infertility, for example, by going through ivf when he has sperm problems, or making sure that he will have progeny when his wife lacks ovaries or uterine function by employing a surrogate gestator or an egg donor. women's resort to art, however, is largely driven by their own desire for children, reflecting personal and cultural narratives of the importance of reproduction for female identity. science not sex becomes the primal scene, with the glare of laboratory lights replacing the darkness of the fallopian tubes. new narratives fit ivf and embryo freezing into old ones of husband and wife having children. surrogate motherhood challenged that narrative because it was so directly an alienation of gestation from rearing and so explicitly for a price (though a diminished gift rationale remained). it devalued the female genetic tie but kept the gestating mother as rearing mother. for women who valued genes over gestation, it was a greater challenge. both areas came to privilege preconception / implantation intent over the later wishes of the gestational surrogate. egg donors also lost out on rearing rights if the recipient's gestation and rearing had been agreed to in advance. with egg donation and surrogacy now widely accepted, egg freezing seems empowering. by shifting or rescheduling genetic motherhood to a later time , it enables women to enter the workforce, establish career, and undergo cancer treatment with less of a chance of missing out on genetic motherhood. on this view, the empowering aspect, though firmly rooted in cultural narratives of the importance of female reproduction, is more important than any accompanying alienation or shift in art business and profit models. the main task is to make freezing as safe and effective as other forms of art, bring the cost down, and ensure that women know what they are getting into. another perspective, while crediting the empowering narrative, will remind women that there are costs here as well, such as the likely low success rate of freezing after 35 and the need to find an experienced program. this translates to storing eggs at the earliest age possible, which alters the passage of women to early adulthood.? it also means resisting a de rigueur attitude to freezing and pro - life efforts to diminish the right to freeze and discard embryos. the trick is to find the sweet spot between these goals. in striking that balance within the prevailing market framework, women may come to view their eggs not only as products of their body essential for their genetic motherhood, but also as products that can be streamed into commerce. this can arise from women who no longer need to store eggs and want to recoup their investment or make a profit. it will also arise in the egg donor market, which despite payment has residues of gift economy. now egg donors will be simply paid for their ovarian bounty, with less of a connection to the recipients they are helping. it may also lead them to negotiate prices pegged to the number of eggs produced. once everyone adjusts, the rough edges may smooth, increasing welfare and flourishing for most participants. as usual, how it is done rather than that it is done will probably turn out to be the key issue. the onslaught of genetic screening now waiting in the wings will be a far more important development for all concerned. mapping egg freezing on to these issues and showing how regulation largely by professional societies emerge with all its flaws will help in devising practices and policies for egg freezing and other innovations in assisted reproduction.

with the development of rapid freezing of human oocytes, many programs have reported ivf success rates comparable to those achieved with fresh eggs and thawed frozen embryos. egg freezing is now gaining professional and regulatory acceptance as a safe and effective technique for women who wish to avoid discarding excess embryos, who face fertility - threatening medical treatments, or who want to preserve their eggs for use when they are better situated to have a family. this article focuses on the uses of and justification for egg freezing, the path to professional acceptance, the variability in success rates, and the controversy over freezing eggs for social rather than medical reasons. it also addresses the emergence of egg banking as a separate sector in the infertility industry, the regulatory issues that it poses, and its effect on egg donation. key here is the legal control of stored eggs by banking women and their options when they wish to dispose of those eggs. the analysis is framed around empowerment and alienation. egg freezing is generally empowering for women, but the donation or sale of unused eggs to infertile women, egg bankers, and researchers also raises issues of alienation.

the human slx1-slx4 structure - selective endonuclease plays a key role in dna repair, homologous recombination, replication fork restart, and telomere maintenance (svendsen and harper, 2010). the genes encoding saccharomyces cerevisiae slx1 and slx4 were discovered in a genetic screen for mutations that are synthetic lethal in the absence of the sgs1 helicase, a protein that is important for genome stability (mullen et al ., 2001). homology searches subsequently identified the slx1 and slx4 genes in higher eukaryotes (andersen et al . , 2009 ; fekairi et al ., 2009 ; muoz et al ., slx1 is an evolutionarily conserved protein that contains an n - terminal giy - yig nuclease domain ( also called uri domain) and a c - terminal zinc - finger domain. giy - yig domains also are present in homing nucleases, the bacterial nucleotide excision - repair nuclease uvrc, and several type ii restriction enzymes (dunin - horkawicz et al ., 2006). the mechanism of substrate binding and cleavage for giy - yig family members has been elucidated by crystallographic studies of protein - dna complexes obtained for two restrictases, namely r.eco29kl (mak et al ., 2010) and hpy188i (sokolowska et al ., 2011). the slx4 subunit of the slx1-slx4 nuclease is thought to provide a scaffold that coordinates the actions of a number of proteins involved in dna processing (cybulski and howlett, 2011). for example, vertebrate slx4 is a large, multi - domain protein that interacts with several dna repair proteins (andersen et al . , 2009 ; fekairi et al ., 2009 ; muoz et al ., 2009 ; salewsky et al ., 2012 ; svendsen et al ., 2009): the n - terminal region of human slx4 binds the msh2-msh3 mismatch - repair complex and xpf - ercc1 nucleotide excision - repair enzyme; and the c - terminal portion of slx4 binds the telomeric proteins trf2 and rap1, the plk1 kinase, and the mus81-eme1 endonuclease. in all organisms studied to date, slx1 binds to the extreme c - terminal region of slx4, which contains an evolutionarily conserved helix - turn - helix motif. interestingly, in vitro studies have shown that slx4 stimulates the endonuclease activities of slx1, mus81-eme1, and xpf - ercc1 (hodskinson et al ., 2014 ; muoz et al ., 2009 ; wyatt et al ., the importance of slx4 is demonstrated by the observation that biallelic mutations in slx4 ( also known as fancp) are associated with the cancer - prone disorder fanconi anemia (bogliolo et al ., 2013 ; kim et al ., 2011 ; stoepker et al ., although the amino acid sequence of slx4 is evolutionarily diverse, the c - terminal region of all slx4 proteins contains a conserved c - terminal domain ( ccd) that underpins the interaction with slx1 and a dna - binding sap domain found in many dna repair proteins (andersen et al . , 2009 ; aravind and koonin, 2000 ; fekairi et al ., 2009 ; muoz et al ., 2009 ; svendsen et al ., , there are few other discernible domains, whereas slx4 proteins from higher eukaryotes ( e.g., worms, flies, and humans) contain one or two copies of a ubz family zinc - finger domain known as ubz4; the mei9 interaction like region (mlr); and a broad - complex, tramtrack, and bric - a - brac (btb) domain (stogios et al ., 2005). the role of slx1-slx4 in dna repair has been studied extensively (cybulski and howlett, 2011 ; sarbajna and west, 2014 ; wyatt and west, 2014). although deletion of slx1 in yeast does not affect the response to dna damage, it has been shown that slx1-slx4 plays a role in maintaining the integrity of ribosomal loci, which contain tandem repeats that frequently lead to replication fork arrest (coulon et al ., 2004). it is possible that slx1-slx4 is involved in the collapse of stalled forks and the resolution of recombination intermediates, such as holliday junctions (hjs), after fork recapture (gritenaite et al ., 2014). in human cells, transient depletion of slx4 leads to an increased sensitivity to alkylating and crosslinking agents, indicating the importance of slx4 for the repair of dna inter - strand crosslinks (icls) and protein - dna adducts. depletion of slx4 also reduces the efficiency of double - strand break repair and leads to genome instability (garner et al ., 2013 ; muoz et al ., 2009 ; sarbajna et al ., 2014 ; svendsen et al ., 2009 ; wechsler et al ., collectively, these observations indicate that slx1 and/or slx4 have relatively well - conserved roles in processing dna intermediates that arise at stalled or collapsed replication forks, particularly when cells are treated with dna - damaging agents that interfere with normal replication fork progression . purified s. cerevisiae slx1-slx4 cleaves various dna substrates in vitro, including splayed - arm structures, model replication forks, 5-flaps, and hjs ( fricke and brill, 2003). for the 5-flap substrates, the major cleavage site lies in the 5 single - stranded arm at the junction between single- and double - stranded dna. whereas slx1 alone possesses weak nuclease activity, purified human slx1-slx4 exhibits related activities and cleaves 5-flaps, 3-flaps, replication forks, and hjs. of particular interest, slx1-slx4 and mus81-eme1 cooperate during hj resolution, with slx1-slx4 performing the initial nick such that mus81-eme1 can resolve the nicked hj without substrate dissociation (wyatt et al ., 2013). although there is a significant amount of functional information available for slx1-slx4 and its associated proteins, structural and mechanistic insights for this enzyme are lacking. here we report the crystal structure of the slx1 nuclease, obtained using candida glabrata slx1 (cg - slx1). the protein forms a compact structure with the giy - yig nuclease and ring - finger domains interacting with each other, and the structural arrangement is reinforced by a long helix. we find that cg - slx1 forms a stable homodimer in the absence of cg - slx4. importantly, the crystal structure of cg - slx1 in complex with cg - slx4, together with biochemical analyses, demonstrate that cg - slx1 homodimerization is mutually exclusive with the formation of a cg - slx1-slx4 heterodimer, revealing a likely regulatory mechanism for slx1 endonuclease activity. to obtain structural information for slx1, we purified c. glabrata slx1 protein (cg - slx1) alone and in complex with the conserved c - terminal domain of slx4 (cg - slx1-slx4). when the enzymatic activities of cg - slx1 and cg - slx1-slx4 were verified using synthetic dna substrates (for sequences, see figures s1a and s1b), we found that cg - slx1 exhibited little or no activity, whereas the cg - slx1-slx4 complex cleaved a diverse set of branched dna structures (figure 1). the observed nuclease activity was inherent to slx1, as substitution of an invariant residue in the active site (slx1) abolished the nuclease activity of cg - slx1-slx4 (figure 1b, bottom). we observed that the hj was the preferred substrate for cg - slx1-slx4, followed by 5-flap, splayed arm, and 3-flap dna structures (figures 1b and 1c). in contrast, the enzyme failed to cleave gapped, nicked, and double- or single - stranded dna substrates. a similar substrate preference was reported for full - length yeast (fricke and brill, 2003) and human (wyatt et al ., 2013) enzymes, although cg - slx1-slx4 was comparatively more active on 3-flaps. enzyme titration experiments with the 5-flap substrate revealed that the primary cleavage product, a nicked or gapped dna duplex, was processed further to generate a shorter dna duplex (figure 1a). however, time - course experiments carried out with limiting amounts of protein revealed that the major reaction was a single nick that converted the 5-flap into a linear duplex (figure 1b). this is consistent with the inability of cg - slx1-slx4 to cleave nicked or gapped dna duplexes when protein is limiting. to gain more insight into the mechanism by which cg - slx1-slx4 cleaves 5-flaps, we prepared two different substrates, each p - labeled on a different oligonucleotide (figures s1c and s1d). time - course experiments revealed two independent cleavage sites: a primary incision that removed the 5-flap, and a less efficient incision reaction that removed the duplex arm. collectively, our provide additional evidence for the promiscuous nature of slx1-slx4, suggesting that this biochemical property has been conserved throughout evolution. we next obtained crystals of cg - slx1, which belonged to the p43212 space group, and diffracted x - rays to 2.34 resolution. the protein was predicted to contain a zinc finger, so diffraction data were collected at the zinc absorbance peak wavelength (1.280), and the structure was solved by zinc single - wavelength anomalous diffraction (sad) (table s1). the asymmetric unit of the crystal contained one slx1 molecule and the structure was refined to an rfree of 25.1% (table s1). fragments of simulated annealing composite omit electron density maps are presented in figure s2a. we noted that the loop regions of the zinc - finger domain had higher b factors and less - well - defined electron density maps, indicating the flexibility of this part of the structure. the cg - slx1 monomer consists of two distinct domains: an n - terminal giy - yig or uri nuclease domain, and a c - terminal zinc - finger domain. in the cg - slx1 structure, these two domains interact with each other and form an oblong molecule approximately 70 long and 3040 wide (figure 2). the overall structure is reinforced by a long helix (helix 6, located between the two domains and comprising residues 176214) that spans the entire molecule and provides a scaffold for the two domains. the central element of the nuclease domain is a sheet consisting of five strands arranged 2-1-3-6-7 (numbered in the amino acid sequence, figure s2c), with strands 2 and 3 oriented antiparallel to 1, 6, and 7 (figure 2). the sheet is flanked by six helices; short helix 1 and long helix 6 are located on one side of the sheet and four helices are positioned on the other side. the 25 region also contains a hairpin formed by strands 4 and 5 (figure 2). comparisons of cg - slx1 structure with related giy - yig nucleases can be found in the supplemental and in figures s2d the active site of cg - slx1 is highly conserved with other giy - yig nucleases (figure s2h), and we therefore propose that the catalytic mechanism of slx1 is identical to that described for hpy188i (sokolowska et al ., 2011). details of the organization of the active site and the catalytic mechanism proposed for cg - slx1 can be found in the supplemental and in figures s2h and s2i. the c - terminal part of the cg - slx1 structure comprises a zinc - finger domain. our structure shows that it is a ring finger closely related to domains found in ubiquitin ligases (supplemental ; figures s2j inspection of the cg - slx1 crystal packing revealed that a 2-fold crystallographic axis generated a very tight protein dimer, with a total buried surface area of 4,729 ( figure 3a). at the dimer interface, helix 2 of one monomer was positioned snugly in a groove located between the nuclease and ring domains of the other subunit. notably, the side chain of arg72, found at the n terminus of helix 2, was inserted into a pocket on the surface of the other subunit, where it interacted with the backbone carbonyls of glu3 and gln5. another prominent interaction that stabilized the homodimer involved tyr86 from the slx1-specific 4-5 hairpin of one subunit and helix 7 from the ring domain of the other subunit. contacts were also made between glu121, tyr122, and tyr86 from one subunit and thr271, ile272, and ile273 of the other. together, these residues formed a network of charged and van der waals interactions through their side chains. two symmetrical copies of this part of the interface were located close to each other and formed a continuous zipper of interdigitated amino acid side chains (figure 3b). the extensive dimerization contacts and large buried surface area prompted us to investigate whether cg - slx1 dimerization occurs in solution. to do this, we used gel filtration coupled to multi - angle light scattering (gf - mals) (figure 3c) and analytical ultracentrifugation (auc) (figure s3a). the molecular weight (mw) of cg - slx1 measured by gf - mals was 80.3 kda and 72.0 kda by auc, suggesting that cg - slx1 exists as a homodimer in solution (calculated mw of 72.0 kda). first, we used gf - mals to determine that the mw of cg - slx4 alone was 19.0 kda. this is in agreement with the theoretical mw of 19.7 kda and indicates that cg - slx4 is monomeric in solution. for cg - slx1-slx4, the measured mw was 57.0 kda (gf - mals) and 47.0 kda (auc). importantly, these data indicate that the cg - slx1-slx4 complex contains one molecule of slx1 and one molecule of slx4 (calculated mw of 57.0 kda). our therefore demonstrate that cg - slx1 alone exists as a stable homodimer, but, in the presence of cg - slx4, interacts with this subunit to form a stable heterodimeric complex. close inspection of the cg - slx1 homodimeric structure showed that the active site of each subunit was partially blocked by the ring domain of the other subunit (figures 3a and s4). indeed, cg - slx1 (figure 1a) as well as the s. cerevisiae (fricke and brill, 2003) and human (muoz et al ., 2009) enzymes are inactive in the absence of slx4. to gain further insight into the cg - slx1 homodimerization, we prepared a series of cg - slx1 variants with substitutions in dimer interface residues. additional gel filtration experiments demonstrated that cg - slx1 homodimerization and cg - slx1-slx4 complex formation are mutually exclusive, and the exchange between the two forms can be promoted by high salt concentration (supplemental ; figures s3d and s3e). in summary, furthermore, they indicate that cg - slx1 dimerization provides a physical block to the active site. thus, these findings provide a potential mechanism by which slx1 nuclease activity is inhibited. nonetheless, cg - slx1-dna interactions can be modeled based on the conservation of the active site between slx1 and giy - yig restrictases, for which high - resolution protein - dna structures are available. to this end , we superimposed the eco29ki structures (protein data bank i d 3nic) on our cg - slx1 structure, which ed in a good fit between dna from the eco29ki structures and the surface of slx1. the modeling revealed that cg - slx1 contains two groups of potential dna - binding residues, all but one of which are located in the nuclease domain (figure 4a). the first group (arg35, arg38, gln39, and gln191) could interact with the non - cleaved dna strand toward its 5 end from the active site. based on the polarity of the dna at the active site, these residues are predicted to contact regions of flap and splayed arm substrates that contain only double - stranded dna (figure 4b). arg35, arg38, and gln39 were all located in the vicinity of the phosphate groups of the modeled dna. sequence alignment of fungal slx1 proteins revealed that arg35 and gln39 are strictly conserved and arg38 is partially conserved (figure s2c). non - conserved gln191 was also located in this region but was positioned farther from the substrate in our cg - slx1-dna model. the second group of predicted dna - binding residues (arg72, gln77, his80, and his84) was located on the opposite face of the active site relative to the first group. these residues are predicted to contact the 5 region of the cleaved strand that exists as single - stranded dna in flap and splayed arm substrates. in the eco29kl - dna structure, the straight dna duplex extended farther away from these residues, suggesting that the single - stranded portion of the splayed arm or 5-flap substrates must be bent to interact with the protein. gln77, his80, and his84 were all located in helix 2 very close to the metal - coordinating active site residue glu79. among fungal slx1 proteins, although his80 is substituted by a tryptophan in most species, his84 is always replaced with a charged residue. interestingly, both arg72 and the region around his80 are buried in the cg - slx1 homodimer, which likely contributes to its inhibition (figure s4a). to verify the importance of the potential dna - binding residues identified in our model , we prepared several cg - slx1 variants in which these residues were individually substituted with alanine. the cg - slx1 mutants were purified in the context of the cg - slx1-slx4 complex and enzyme activity was determined using the 5-flap substrate (figure 4c). these mutants showed either a complete loss of nuclease activity (r35a and q39a) or substantially impaired activity (r38a, r72a, q77a, q191a, h80, and h84). we also tested the ability of these slx1 variants to bind the 5-flap substrate using electrophoretic mobility shift assays (emsas) (figure 4d). cg - slx1 alone did not exhibit dna - binding properties, while cg - slx1-slx4 formed a protein - dna complex with reduced mobility. at high protein concentrations, two protein - dna complexes were observed, possibly indicating the binding of two slx1 molecules to dna. the r38a and q39a mutants showed slightly stronger substrate binding compared to the wild - type protein, despite exhibiting severe catalytic defects. importantly, arg38 and gln39 are located close to the active site and gln39 forms a hydrogen bond with active site residue arg36. its greatly reduced activity suggests that arg72, which is located in the vicinity of the scissile phosphate, has a more important role in aligning the substrate for cleavage than enhancing substrate affinity. the remaining mutants, namely r35a, q77a, h80a, h84a, and q191a, all displayed defects in dna binding. his84 is located in helix 2, which we postulate to participate in binding the single - stranded portion of the 5-flap dna. this further underscores the importance of helix 2 in the dna - binding interface. we have therefore identified two regions of cg - slx1 that are important for protein - dna interactions: one predicted to bind double - stranded portion of the dna, and the other potentially interacting with the single - stranded flap. our initial crystallization trials with cg - slx1-slx4 did not yield any crystals, so to gain further insight into cg - slx1-slx4 interaction, we performed additional deletion studies. based on bioinformatic predictions and limited proteolysis experiments, we designed four truncated forms of slx4, comprising residues 557685 (cg - slx4), 608685 (cg - slx4), 557698 (cg - slx4), or 647726 (cg - slx4) (figure all of these fragments were soluble when expressed in escherichia coli ( figure s5b), but only the ccd3 variant co - purified with cg - slx1 (figures s5c and s5d). when we analyzed the activity of cg - slx1-slx4 on the 5-flap dna, we found that it displayed slightly less activity than cg - slx1-slx4 and that the cleavage sites for both ccd variants were similar (figure s5e). they belonged to the p63 space group and diffracted x - rays to 1.8 (table s1). the structure was solved by molecular replacement using the cg - slx1 model and the structure of cg - slx4 was traced manually (figures 5 and s2b). the overall conformation of cg - slx1 domains did not change significantly upon slx4 binding, although the location of the ring domain changed slightly (figures s5f and s5 g). when cg - slx1 and cg - slx1-slx4 were superimposed using the giy - yig domains, the position of the ring domain residues differed by up to 3.5. the 4-5 hairpin was not formed in the cg - slx1-slx4 complex and this part of the structure adopted a different conformation (figure 5a). its closest structural homologs are ff domains mediating protein - protein interactions (bedford and leder, 1999). for example , prp40 ff1 domain (pdb i d 2b7e) can be superimposed on cg - slx4 with an rmsd of 1.6 over 37 c atoms. notably, however, the characteristic phenylalanine residues of the ff domain are not conserved in cg - slx4. cg - slx4 was positioned in a cleft between the nuclease and ring domains, and most of the interactions between the cg - slx1 and cg - slx4 occurred through hydrophobic contacts (figure 5b). in particular, the second short helix of cg - slx4 was placed snugly in a hydrophobic groove on the cg - slx1 surface. the groove lined up with the side chains of cg - slx1 phe4 (nuclease domain) and tyr257, ile292, and ile273 (ring domain). the side chain of cg - slx4 phe681, which is conserved in most fungal slx4 sequences (figure s5a) and is located in the second short helix, was inserted into a pocket on the cg - slx1 surface. the hydrophobic interactions were reinforced further by stacking of cg - slx1 tyr257 with cg - slx4 tyr679. in addition, the backbone carbonyl of this tyrosine formed a hydrogen bond with the n1 of the conserved trp288 in cg - slx1. additional residues at the cg - slx1-slx4 interface included cg - slx1 ile73, tyr122, and tyr125, all of which interacted with the c terminus of the last helix of cg - slx4 and the following loop (residues 714720). the importance of the latter interaction was confirmed by the lack of binding between cg - slx1 and slx4, in which the c - terminal helix of the ccd is absent. in murine slx4, the c1536r mutation abolished its interaction with slx1 (castor et al ., 2013). this cysteine corresponds to cg - slx4 gly716 located at the c terminus of the last helix of cg - slx4. insertion in this position of an arginine residue with a large side chain would lead to steric clashes with cg - slx1, explaining the effect of c1536r mutation. importantly, the position of cg - slx4 in the slx1-slx4 heterodimer overlapped with the position of one of the slx1 monomers in the cg - slx1 homodimer (figures s5f and s5 g). in fact, several residues, including cg - slx1 tyr122 and ile273, participated in both cg - slx1 homodimerization and cg - slx1-slx4 heterodimerization, thus explaining why the formation of cg - slx1 homodimeric and cg - slx1-slx4 heterodimeric complexes are mutually exclusive events. slx4 was located away from the predicted dna - binding interface of cg - slx1 and is not expected to form contacts with the substrate (figure s4b). in , the studies presented here provide the first structural information for slx1 and its interaction with slx4. structure - selective endonucleases pose an inherent threat to genome integrity because broken dna ends can facilitate chromosome rearrangements and genome alterations that are potentially tumorigenic. it is therefore crucial to keep these endonucleases tightly regulated to ensure cleavage of the correct dna substrate at an appropriate time in the cell cycle. recent studies have revealed that these regulatory mechanisms operate at various levels: protein expression (courcelle et al ., 2001), post - translation modification (mus81-eme1/mms4, yen1) (blanco et al ., 2014 ; matos et al ., 2011, 2013), nuclear localization (gen1) (chan and west, 2014), conformational changes (fen1) (kim et al ., 2001 ; storici et al ., 2002), and protein - protein interactions (e.g., xpg, xpf - ercc1, and slx - mus) (arajo et al ., 2001 ; li et al ., 1994 ; our data suggest a mechanism of slx1 regulation through inhibitory homodimerization that would keep slx1, a promiscuous and potentially dangerous endonuclease, latent and ensure that its activity is tightly regulated in cells . purified cg - slx1, at a concentration of 10 mg / ml, was subjected to crystallization screens at 18c using the sitting - drop vapor diffusion method . prior to crystallization, the protein was dialyzed against buffer containing 20 mm hepes - naoh ( ph 7.5), 350 mm nacl, and 1 mm dtt. crystals were obtained with 1.4 m sodium citrate tribasic dihydrate and 0.1 m hepes - naoh (ph 7.5). purified cg - slx1-slx4, at a concentration of 10 mg / ml, was subjected to crystallization screens at room temperature using the sitting - drop vapor diffusion method. prior to crystallization, the protein was dialyzed against buffer containing 20 mm hepes - naoh (ph 7.5), 150 mm nacl, and 1 mm dtt. crystals were obtained with 0.2 m magnesium chloride hexahydrate, 0.1 m bis tris (ph 6.5), and 25% (v / v) peg 3350 in the presence of a splayed arm dna substrate with 15-bp double - stranded portion and 5-nt single - stranded overhangs. x - ray diffraction data for cg - slx1 were collected at beamline 14.1 at berliner elektronenspeicherring - gesellschaft fr synchrotronstrahlung (bessy) (mueller et al ., 2012), and data for cg - slx1-slx4 were collected at beamline id29 at european synchrotron radiation facility (esrf). data for the phasing and refinement of cg - slx1 structure were collected at 1.280 wavelength. diffraction data used for refinement of the cg - slx1-slx4 structure were collected at 0.97626. phases for cg - slx1 were determined using single - wavelength anomalous diffraction in autosol module in phenix (adams et al ., 2010), using data collected at zn peak wavelength (1.280). phases for cg - slx1-slx4 were determined by molecular replacement using phaser - mr module in phenix (adams et al ., 2010). the structure of cg - slx1 was used as the starting model for molecular replacement. interactive model building was performed in coot (emsley et al ., 2010) and refinement with phenix with r - free, calculated with 5% of unique reflections. the structures of cg - slx1 and cg - slx1-slx4 were refined with 95.7% and 99.7% residues in the favored region of the ramachandran plot, respectively. structure validation was carried out using molprobity analysis (chen et al ., 2010). structural analyses, including superpositions, and structural figures were prepared in pymol (http://www.pymol.org). simulated annealing composite omit maps were calculated in cns 1.3 (brnger et al ., 1998). description of protein purification, oligomeric state analysis, nuclease assays, and emsa can be found in the supplemental experimental procedures.

summarythe slx1-slx4 endonuclease required for homologous recombination and dna repair in eukaryotic cells cleaves a variety of branched dna structures. the nuclease subunit slx1 is activated by association with a scaffolding protein slx4. at the present time, little is known about the structure of slx1-slx4 or its mechanism of action. here , we report the structural insights into slx1-slx4 by detailing the crystal structure of candida glabrata (cg) slx1 alone and in combination with the c - terminal region of slx4. the structure of slx1 reveals a compact arrangement of the giy - yig nuclease and ring domains, which is reinforced by a long helix. slx1 forms a stable homodimer that blocks its active site. slx1-slx4 interaction is mutually exclusive with slx1 homodimerization, suggesting a mechanism for slx1 activation by slx4.

pelvic fractures comprise < 0.2 % of all pediatric fractures but constitute up to 5 % of admissions to level i pediatric trauma centers. like adults, pediatric pelvic fractures are associated with high - energy trauma and injury to other systems, leading to an increased incidence of complication and mortality. due to the pliable nature of the child s skeleton, severe soft tissue trauma may occur without producing skeletal injury. thus, a child presenting with a pelvic fracture should be suspected of having a multiorgan injury with an increased potential for injury, complications, and death. during the transition period between childhood and adulthood, known as adolescence, children gain size and strength, making their body increasingly resistant to extrinsic injury. however, previous studies on pediatric pelvic fractures analyzed the pediatric population as a single group of both children and adolescents. biological, psychological, social, and environmental changes influence the onset and termination of adolescence. physiologic changes that occur during adolescence, such as increase in muscle size and bone mass, doubling in heart size and lung vital capacity, and rise in blood pressure, blood volume, and hematocrit (particularly in boys), may lead to a theoretical protective effect against trauma. similar to growing children, adolescents have an osteoblast: osteoclast activity ratio > 1, which increases their capacity to heal fractures. these physiological advantages, together with the increase in distance between internal organs, weight to surface area ratio, and cardiovascular reserve, may make adolescents particularly resilient to traumatic injury. the purpose of this study is to examine whether adolescents with pelvic fracture have different complication and mortality rates compared to younger children and adults. we identified the study population by means of the national trauma data bank (ntdb version 7.1). this ntdb version contained over 2.7 million cases from over 900 us trauma centers between the years 2002 and 2006. the data were imported and merged into a single dataset from the 13 ntdb files using sas version 9.2 (sas institute, cary, nc). for the purpose of our study, all burn or penetrating injuries were excluded, which reduced the group to 1.7 million cases. if a patient s multiple icd-9 diagnosis codes contained at least one of the following codes, the patient was considered to have a pelvic fracture: 808.2, 808.3, 808.4, 808.41, 808.42, 808.43, 808.49, 808.5, 808.51, 808.52, 808.53, 808.59, 808.8, 808.9. acetabular fractures were excluded from the study. those entries without a pelvic fracture were removed, giving a total of 54,459 cases of pelvic fractures. finally, all adults aged 55 years and older were removed from the study, yielding a final study population of 37,784. this final study population was subdivided into our three groups of interest: children (younger than 13 years old), adolescents (aged 1317 years), and adults (aged 1854 years). severe complication was defined as having at least one of the following complications recorded: renal failure, pneumonia, bacteremia, acute respiratory distress syndrome (ards), deep vein thrombosis (dvt), or a pulmonary embolism. prehospital risk factors such as sex, race, age, arrival in shock (systolic blood pressure < 90 mmhg), injury severity score (iss), head injury, and mechanism of injury were also analyzed to determine their association with the main outcomes. descriptive statistics were performed on the entire study population (n = 37,784). to determine associations between risk factors and the main outcomes of interest, bivariate analysis was conducted between each prehospital risk factor and two main outcomes (mortality and severe complication). for this and subsequent assessment, open fractures (only 4.2 % of pelvic fractures and highly variable in presentation and complication profile) and unknown mechanisms of injury were excluded, yielding a study group of 24,684. a subpopulation of severe pelvic injuries was also created to determine if any pediatric subgroup has better outcomes. for this population, an abbreviated injury scale (ais) score of <3, signifying minor and moderate injuries, and all patients with lower extremity fractures besides pelvic fractures were excluded. this created a study subgroup of 2,487 patients with isolated severe pelvic fractures. for both the main study population and the subgroup of severe pelvic injury, prehospital risk factors were determined to be significant using the mantel this method produced crude odds ratios (ors) and 95 % confidence intervals (cis), and those variables of significance were included for the multivariate analysis. logistic regression analyses including those variables of significance were performed to determine the association between the prehospital risk factors and the two main outcomes. each model included a specific age group (children, adolescent, and adult) as compared to all other ages in order to assess the importance of age and outcome after sustaining a pelvic fracture. the hosmer lemeshow goodness - of - fit test was performed on each model to determine whether or not the observed event rates matched the expected event rates. descriptive statistics were performed on the entire study population (n = 37,784). to determine associations between risk factors and the main outcomes of interest, bivariate analysis was conducted between each prehospital risk factor and two main outcomes (mortality and severe complication). for this and subsequent assessment, open fractures (only 4.2 % of pelvic fractures and highly variable in presentation and complication profile) and unknown mechanisms of injury were excluded, yielding a study group of 24,684. a subpopulation of severe pelvic injuries was also created to determine if any pediatric subgroup has better outcomes. for this population, an abbreviated injury scale (ais) score of <3, signifying minor and moderate injuries, and all patients with lower extremity fractures besides pelvic fractures were excluded. this created a study subgroup of 2,487 patients with isolated severe pelvic fractures. for both the main study population and the subgroup of severe pelvic injury, prehospital risk factors were determined to be significant using the mantel this method produced crude odds ratios (ors) and 95 % confidence intervals (cis), and those variables of significance were included for the multivariate analysis. logistic regression analyses including those variables of significance were performed to determine the association between the prehospital risk factors and the two main outcomes. each model included a specific age group (children, adolescent, and adult) as compared to all other ages in order to assess the importance of age and outcome after sustaining a pelvic fracture. the hosmer lemeshow goodness - of - fit test was performed on each model to determine whether or not the observed event rates matched the expected event rates. our study s overall incidence rate of pelvic fractures was 26 cases per 10,000 trauma admissions per year for adults, 4 cases per 10,000 trauma admissions per year for adolescents, and 5 cases per 10,000 trauma admissions per year for children. severe pelvic fractures had an incidence rate of 3.5 cases per 10,000 per year for adults, 0.47 cases per 10,000 per year for adolescents, and 0.63 cases per 10,000 per year for children. descriptive statistics of demographics, injury mechanism, injury severity, treatment, and complications are presented in table 1. after sustaining a pelvic fracture, survival was 91.4 % for the total population. when separated by age group, adolescents appeared to have the best survival statistics (93.2 %), while children had the worst (89.8 %). adolescents had the lowest percentage of severe complication (11.4 %), children had a slightly higher percentage (13.0 %), and adults had the highest percentage (15.7 %).table 1frequency distribution of characteristics for the entire population of pelvic fractures, n = 37,784variableage grouptotal, n (%) children (< 13 years), n (%), n = 5,325adolescents (1317 years), n (%), n = 4,052adults (1854 years), n (%), n = 28,407gender male2,349 (44.8)1,888 (46.7)17,393 (61.3)21,630 (57.4) female2,897 (55.2)2,157 (53.3)10,983 (38.7)16,037 (42.6)race caucasian3,570 (72.3)2,745 (73.4)18,301 (69.9)24,616 (70.7) african american474 (9.6)341 (9.1)2,717 (10.4)3,532 (10.1) asian / pacific islander73 (1.5)54 (1.4)504 (1.9)631 (1.8) hispanic490 (9.9)394 (10.6)3002 (11.5)3,886 (11.2) native american31 (0.6)31 (0.9)201 (0.8)263 (0.8) other299 (6.1)173 (4.6)1,440 (5.5)1,912 (5.4)survival survived4,766 (89.8)3,753 (93.2)25,853 (91.5)34,372 (91.4) died542 (10.2)273 (6.8)2,404 (8.5)3,219 (8.6)severe complication yes693 (13.0)462 (11.4)4,465 (15.7)5,620 (14.9) no4,632 (87.0)3,590 (88.6)23,942 (84.3)32,164 (85.1)fracture type open183 (3.4)96 (2.4)1,316 (4.6)1,595 (4.2) closed5,142 (96.6)3,956 (97.6)27,078 (95.4)36,176 (95.8)fracture location ilium703 (13.7)622 (15.7)3,797 (13.9)5,122 (14.1) ischium162 (3.1)153 (3.9)1,151 (4.2)1,466 (4.0) pubis2,615 (42.0)1,632 (41.2)11,281 (41.4)15,078 (41.5) multiple330 (6.4)220 (5.5)1,799 (6.6)2,349 (6.5) unspecified673 (13.1)342 (8.6)2,328 (8.6)3,343 (9.2) other1,120 (21.7)996 (25.1)6,878 (25.3)8,994 (24.7)systolic blood pressure (mmhg) < 90878 (16.5)524 (12.9)4,615 (16.3)6,017 (15.9) 901392,869 (53.9)2,680 (66.2)16,704 (58.8)22,253 (58.9) 140 + 1,578 (29.6)848 (20.9)7,088 (24.9)9,514 (25.2)moi motor vehicle1,329 (24.9)2,224 (54.9)11,520 (40.6)15,073 (39.9) motorcycle119 (2.2)91 (2.3)2,674 (9.4)2,884 (7.6) pedestrian170 (3.2)102 (2.5)566 (1.9)838 (2.2) crush93 (1.8)31 (0.7)732 (2.6)856 (2.3) high fall592 (11.1)108 (2.6)2,680 (9.5)3,380 (9.0) low fall1,216 (22.8)63 (1.6)1,393 (4.9)2,672 (7.1) unknown1,806 (34.0)1,433 (35.4)8,842 (31.1)12,081 (31.9)ais of pelvis none3,323 (62.4)2,660 (65.6)19,331 (68.1)25,314 (67.0) 19 (0.2)11 (0.3)96 (0.3)116 (0.3) 21,313 (24.6)869 (21.5)5,153 (18.2)7,335 (19.4) 3610 (11.5)450 (11.1)3,129 (11.0)4,189 (11.1) 453 (1.0)45 (1.1)522 (1.8)620 (1.6) 517 (0.3)17 (0.4)176 (0.6)210 (0.6) 60 (0.0)0 (0.0)0 (0.0)0 (0.0)iss < 254,338 (81.5)2,816 (69.5)19,721 (69.4)26,875 (71.1) 25987 (18.5)1,236 (30.5)8,686 (30.6)10,909 (28.9)pneumonia yes102 (1.9)125 (3.1)1,232 (4.3)1,459 (3.9) no5,223 (98.1)3,927 (96.9)27,175 (95.7)36,325 (96.1)acute respiratory stress syndrome yes61 (1.1)55 (1.4)573 (2.0)689 (1.8) no5,264 (98.9)3,997 (98.6)27,834 (98.0)37,095 (98.2)deep vein thrombosis yes27 (0.5)21 (0.5)503 (1.8)551 (1.5) no5,298 (99.5)4,031 (99.5)27,904 (98.2)37,233 (98.5)bacteremia yes10 (0.2)26 (0.6)196 (0.7)232 (0.6) no5,315 (99.8)4,026 (99.4)28,211 (99.3)37,501 (99.4)renal failure yes32 (0.6)15 (0.4)225 (0.8)272 (0.7) no5,293 (99.4)4,037 (99.6)28,182 (99.2)37,512 (99.3)pulmonary embolism yes15 (0.3)5 (0.1)160 (0.6)180 (0.4) no5,310 (99.7)4,047 (99.9)28,247 (99.4)37,604 (99.6)icu stay < 1 week4,890 (91.8)3,545 (87.5)24,086 (84.8)32,521 (86.1) 1 week435 (8.2)507 (12.5)4,321 (15.2)5,263 (13.9)hospital stay < 1 week3,727 (70.0)2,406 (59.4)13,778 (48.5)19,911 (52.7) 1 week1,598 (30.0)1,646 (40.6)14,629 (51.5)17,873 (47.3)major procedure crif1 (0.02)2 (0.05)20 (0.07)23 (0.08) orif245 (4.62)395 (9.71)4,691 (16.47)5,331 (14.1) closed dislocation reduction7 (0.13)19 (0.47)117 (0.41)143 (0.39) open dislocation reduction12 (0.23)15 (0.37)127 (0.45)154 (0.43) no major procedure5,060 (95.0)3,621 (89.4)23,452 (82.6)32,133 (85.0)moi mechanism of injury, ais abbreviated injury scale, iss injury severity score, icu intensive care unit, crif closed reduction internal fixation, orif open reduction internal fixation frequency distribution of characteristics for the entire population of pelvic fractures, n = 37,784 moi mechanism of injury, ais abbreviated injury scale, iss injury severity score, icu intensive care unit, crif closed reduction internal fixation, orif open reduction internal fixation most of the pelvic fractures were closed (95.8 %). pubic rami fractures consistently had the highest prevalence across all age groups (41.5 %). the most common known mechanism of injury was motor vehicle accident (39.9 %), followed by high - energy fall (9.0 %) and motorcycle accident (7.6 %). most patients had an iss < 25 (71.1 %). when divided by age, more children had an iss < 25 (81.5 %) than adolescents (69.5 %) or adults (69.4 %). adolescents also had less reports of hypovolemic shock on arrival (systolic pressures below 90 mmhg) (12.9 %) than children (16.5 %) and adults (16.3 %) who had comparable numbers. for this assessment, open fractures and unknown mechanisms of injury were excluded, yielding a study group of 24,684. the of the bivariate analysis (reporting crude ors and 95 % cis) for this population are depicted in table 2. as compared to the rest of the population , children had a small increase in odds of death (or 1.27, 95 % ci 1.111.44), but a slight decrease in odds of severe complication (or 0.83, 95 % ci 0.750.93). adolescents had decreases in both odds of death (or 0.85, 95 % ci 0.721.00) and severe complication (or 0.78, 95 % ci 0.690.89). adults had a slight decrease in odds of death (or 0.92, 95 % ci 0.821.02), but a small increase in odds of severe complication (or 1.27, 95 % ci 1.161.39). individuals suffering from hypovolemic shock had substantially increased odds of death (or 5.89, 95 % ci 5.326.51) and severe complication (or 3.62, 95 % ci 3.333.93). sustaining a head injury also increased the odds of death (or 2.96, 95 % ci 2.593.39) and complication (or 3.23, 95 % ci 2.903.61). an iss 25 greatly increased the odds of death (or 11.1, 95 % ci 9.9212.5) and complication (or 9.19, 95 % ci 8.489.97). motor vehicle accidents caused the largest increase in odds of death (or 1.46, 95 % ci 1.321.62) and severe complication (or 1.53, 95 % ci 1.421.65).table 2bivariate analysis for the entire population of closed pelvic fractures comparing predictors upon arrival to the emergency department with the outcomes death and severe complication (odds ratios and 95 % confidence intervals), n = 24,684predictorsoutcomedeathsevere complicationmale1.24 (1.131.37)1.41 (1.311.52)not caucasian1.14 (1.041.26)1.00 (0.921.08)pediatric (< 18 years)1.09 (0.981.21)0.79 (0.720.86)children (< 13 years)1.27 (1.111.44)0.83 (0.750.93)adolescent (1317 years)0.85 (0.721.00)0.78 (0.690.89)adults (1854 years)0.92 (0.821.02)1.27 (1.161.39)hypovolemic shock5.89 (5.326.51)3.62 (3.333.93)head injury2.96 (2.593.39)3.23 (2.903.61)iss 2511.1 (9.9212.5)9.19 (8.489.97)motor vehicle accident1.46 (1.321.62)1.53 (1.421.65)motorcycle accident1.34 (1.161.54)1.34 (1.201.49)fall from height0.61 (0.520.72)0.61 (0.540.69)low - energy fall0.41 (0.330.51)0.36 (0.300.43)crush0.43 (0.290.63)0.66 (0.520.84)pedestrian versus auto1.40 (1.111.77)1.13 (0.931.38)iss injury severity scoresevere complication is defined as having one or more of the following during hospital course: pneumonia, bacteremia, deep vein thrombosis, pulmonary embolism, renal failure, acute respiratory distress syndrome, or death bivariate analysis for the entire population of closed pelvic fractures comparing predictors upon arrival to the emergency department with the outcomes death and severe complication (odds ratios and 95 % confidence intervals), n = 24,684 iss injury severity score severe complication is defined as having one or more of the following during hospital course: pneumonia, bacteremia, deep vein thrombosis, pulmonary embolism, renal failure, acute respiratory distress syndrome, or death variables that were found to be significant in the bivariate analysis were included in the final multiple logistic regression models for death and severe complication as outcomes. adjusted ors and 95 % cis are presented in table 3. when controlling for mechanism of injury and other prehospital conditions, children had more than twice the odds of death (or 2.29, 95 % ci 1.962.67) and increased odds of severe complication (or 1.36, 95 % ci 1.201.55) relative to the adult group after sustaining a pelvic fracture. adolescents, on the other hand, had a slight decrease in odds of death (or 0.89, 95 % ci 0.741.06) and lower odds of severe complication (or 0.70, 95 % ci 0.610.81) relative to the adult group after sustaining a pelvic fracture.table 3multivariate logistic regression analysis for the entire population of closed pelvic fractures using predictors upon arrival to the emergency department with the outcomes death and severe complication (odds ratios and 95 % confidence intervals)model predictorsoutcomedeath, n = 24,684severe complication, n = 22,846children (< 13 years)2.29 (1.962.67)1.36 (1.201.55)adolescents (1317 years)0.89 (0.741.06)0.70 (0.610.81)adults (1854 years)referentreferentmale1.19 (1.071.33)1.38 (1.261.50)not caucasian1.00 (0.901.12)hypovolemic shock4.52 (4.055.05)2.81 (2.563.08)head injury1.60 (1.371.86)1.70 (1.501.92)iss 259.15 (8.0710.4)7.41 (6.798.09)motor vehicle accident3.98 (0.3940.2)1.13 (0.921.41)motorcycle accident4.61 (0.4646.8)1.18 (0.921.51)fall from height3.27 (0.3233.1)0.80 (0.631.03)low - energy fall3.18 (0.3231.7)0.77 (0.581.02)pedestrian versus auto4.50 (0.4544.8)crush2.42 (0.2325.3)0.95 (0.691.34)hosmer lemeshow0.00690.17bold values signify 95 % confidence intervals that are completely greater than 1, indicating a significant positive associationiss injury severity scoresevere complication is defined as having one or more of the following during hospital course: pneumonia, bacteremia, deep vein thrombosis, pulmonary embolism, renal failure, acute respiratory distress syndrome, or deathhosmer lemeshow p - value given for the model s goodness of fit instead of odds ratio and 95 % confidence interval multivariate logistic regression analysis for the entire population of closed pelvic fractures using predictors upon arrival to the emergency department with the outcomes death and severe complication (odds ratios and 95 % confidence intervals) bold values signify 95 % confidence intervals that are completely greater than 1, indicating a significant positive association iss injury severity score severe complication is defined as having one or more of the following during hospital course: pneumonia, bacteremia, deep vein thrombosis, pulmonary embolism, renal failure, acute respiratory distress syndrome, or death hosmer lemeshow p - value given for the model s goodness of fit instead of odds ratio and 95 % confidence interval a subgroup analysis was performed for patients determined to have a severe, isolated, closed pelvic injury (ais 3). table 4 demonstrates the descriptive statistics for this subpopulation (n = 2,487). adolescents (n = 255) still appeared to have a lower incidence of mortality (3.1 %) and lower incidence of a severe complication (8.2 %) compared to adults (n = 1,886) and children (n = 346). adolescents contributed the most to motor vehicle accident (89.4 %), which was the most common mechanism of injury in the severe pelvic fracture group. similar to the whole population, the severe pelvic injury group demonstrated more children with an iss < 25 (82.1 %). for cases of hypovolemic shock on arrival, adolescents had the fewest reports of systolic pressures below 90 mmhg (6.7 %), while children (15.9 %) had the largest contribution. children had the lowest incidence of hospital stay and intensive care unit (icu) admission, while adolescents and adults were relatively comparable.table 4frequency distribution of characteristics for individuals with isolated, closed, severe pelvic fractures (ais3), n = 2,487variableage grouptotal, n (%) children (< 13 years), n (%), n = 346adolescents (1317 years), n (%), n = 255adults (1854 years), n (%), n = 1,886gender male127 (38.7)92 (36.2)1,109 (58.9)1,328 (53.8) female201 (61.3)162 (63.8)775 (41.1)1,138 (46.2)race caucasian235 (77.1)168 (74.3)1,208 (73.0)1,611 (73.7) african american31 (10.2)30 (13.3)170 (10.3)231 (10.6) asian / pacific islander4 (1.3)1 (0.4)24 (1.5)29 (1.3) hispanic29 (9.5)20 (8.9)200 (12.1)249 (11.4) native american1 (0.3)2 (0.9)7 (0.4)10 (0.5) other5 (1.6)5 (2.2)46 (2.7)56 (2.5)survival survived321 (92.8)247 (96.9)1,774 (94.1)2,342 (94.2) died25 (7.2)8 (3.1)112 (5.9)145 (5.8)severe complication yes32 (9.3)21 (8.2)209 (11.1)262 (10.5) no314 (90.8)234 (91.8)1,677 (88.9)2,225 (89.5)fracture location ilium41 (11.9)47 (18.4)228 (12.1)316 (12.7) ischium7 (2.0)7 (2.8)53 (2.8)67 (2.7) pubis222 (64.2)143 (56.1)1,059 (56.2)1,424 (57.3) multiple31 (9.0)30 (11.8)277 (14.7)338 (13.6) unspecified10 (2.9)4 (1.6)70 (3.7)84 (3.4) other35 (10.0)24 (9.3)199 (10.5)258 (10.3)systolic blood pressure (mmhg) < 9055 (15.9)17 (6.7)176 (9.3)248 (10.0) 90139173 (50.0)178 (69.8)1,197 (63.5)1,548 (62.2) 140 + 118 (34.1)60 (23.5)513 (27.2)691 (27.8)moi motor vehicle127 (36.7)228 (89.4)1,094 (58.0)1,449 (58.3) motorcycle17 (4.9)6 (2.4)196 (10.4)219 (8.8) pedestrian17 (4.9)5 (2.0)70 (3.7)92 (3.7) crush13 (3.8)5 (2.0)101 (5.4)119 (4.8) high fall52 (15.0)4 (1.5)248 (13.2)304 (12.2) low fall120 (34.7)7 (2.7)177 (9.3)304 (12.2)ais of pelvis 3321 (92.7)231 (90.6)1,606 (85.2)2,158 (86.8) 420 (5.8)17 (6.6)225 (11.9)262 (10.5) 55 (1.5)7 (2.8)55 (2.9)67 (2.7)iss < 25284 (82.1)169 (66.3)1,308 (69.4)1,761 (70.8) 2562 (17.9)86 (33.7)578 (30.6)726 (29.2)pneumonia yes3 (0.9)12 (4.7)53 (2.8)68 (2.7) no343 (99.1)243 (95.3)1,833 (97.2)2,419 (97.3)acute respiratory stress syndrome yes1 (0.3)1 (0.4)28 (1.5)30 (1.2) no345 (99.7)254 (99.6)1,858 (98.5)2,457 (98.8)deep vein thrombosis yes0 (0.0)1 (0.4)19 (1.0)20 (0.8) no346 (100.0)254 (99.6)1,867 (99.0)2,467 (99.2)bacteremia yes1 (0.3)1 (0.4)6 (0.3)8 (0.3) no345 (99.7)254 (99.6)1,880 (99.7)2,479 (99.7)renal failure yes2 (0.6)1 (0.4)7 (0.4)10 (0.4) no344 (99.4)254 (99.6)1,879 (99.6)2,477 (99.6)pulmonary embolism yes1 (0.3)0 (0.0)7 (0.4)8 (0.3) no345 (99.7)255 (100.0)1,879 (99.6)2,479 (99.7)icu stay < 1 week326 (94.2)224 (87.8)1,676 (88.9)2,226 (89.5) 1 week20 (5.8)31 (12.2)210 (11.1)261 (10.5)hospital stay < 1 week256 (74.0)151 (59.2)1,011 (53.6)1,418 (57.0) 1 week90 (26.0)104 (40.8)875 (46.4)1,069 (43.0)major procedure crif0 (0.0)0 (0.0)1 (0.1)1 (0.1) orif31 (9.0)45 (17.6)501 (26.5)577 closed dislocation reduction0 (0.0)2 (0.8)7 (0.4)9 (0.4) open dislocation reduction0 (0.0)0 (0.0)13 (0.7)13 (0.5) no major procedure315 (91.0)208 (81.6)1,364 (72.3)1,887 (75.9)moi mechanism of injury, ais abbreviated injury scale, iss injury severity score, icu intensive care unit, crif closed reduction internal fixation, orif open reduction internal fixation frequency distribution of characteristics for individuals with isolated, closed, severe pelvic fractures (ais3), n = 2,487 moi mechanism of injury, ais abbreviated injury scale, iss injury severity score, icu intensive care unit, crif closed reduction internal fixation, orif open reduction internal fixation similar to what was done with the whole population, bivariate analysis was also performed on the severe pelvic fracture subpopulation (table 5). as compared to the adults, children had higher odds of death but lower odds of severe complication. however, adolescents had a crude decrease in odds of death and severe complication after sustaining a pelvic fracture compared to the adults. individuals suffering from hypovolemic shock, head injury, and iss 25 had an increase in odds of death and severe complications. motor vehicle accidents caused the largest increase in odds of death (or 1.58, 95 % ci 1.102.26), while motorcycle accidents accounted for the largest increase in severe complication (or 1.58, 95 % ci 1.062.34).table 5bivariate analysis for isolated closed pelvic fractures with an associated ais 3, comparing predictors upon arrival to the emergency department with the outcomes death and severe complication (odds ratios and 95 % confidence intervals)predictorsoutcomedeath, n = 2,487severe complication, n = 2,487male1.11 (0.791.56)1.60 (1.222.08)not caucasian1.53 (1.092.15)1.26 (0.971.64)pediatric (< 18 years)0.92 (0.621.37)0.78 (0.571.06)children (< 13 years)1.31 (0.842.05)0.85 (0.571.25)adolescent (1317 years)0.50 (0.241.02)0.74 (0.471.18)adults (1854 years)1.09 (0.731.62)1.29 (0.941.77)hypovolemic shock13.0 (9.0618.6)7.35 (5.449.93)head injury4.41 (2.946.60)5.91 (4.288.16)iss 2527.5 (9.9212.5)14.9 (10.720.7)motor vehicle accident1.58 (1.102.26)1.32 (1.011.72)motorcycle accident1.62 (0.982.67)1.58 (1.062.34)fall from height0.63 (0.341.16)0.74 (0.481.13)low - energy fall0.095 (0.0230.38)0.35 (0.200.61)crush0.85 (0.371.97)0.95 (0.521.75)pedestrian versus auto1.33 (0.602.93)1.33 (0.602.93)bold values signify 95 % confidence intervals that are completely greater than 1, indicating a significant positive associationiss injury severity scoresevere complication is defined as having one or more of the following during hospital course: pneumonia, bacteremia, deep vein thrombosis, pulmonary embolism, renal failure, acute respiratory distress syndrome, or death bivariate analysis for isolated closed pelvic fractures with an associated ais 3, comparing predictors upon arrival to the emergency department with the outcomes death and severe complication (odds ratios and 95 % confidence intervals) bold values signify 95 % confidence intervals that are completely greater than 1, indicating a significant positive association iss injury severity score severe complication is defined as having one or more of the following during hospital course: pneumonia, bacteremia, deep vein thrombosis, pulmonary embolism, renal failure, acute respiratory distress syndrome, or death variables that were found to be significant in the bivariate analysis were included in the final multiple logistic regression models for death and severe complication as outcomes (table 6). when controlling for mechanism of injury and other prehospital conditions and using the adult group as a referent, children had nearly double the odds of death after sustaining a severe pelvic fracture, whereas adolescents had a large decrease in odds of death (or 0.40, 95 % ci 0.180.91). compared to adults, children had potentially increased odds of severe complication after sustaining a severe pelvic fracture (or 1.07, 95 % ci 0.651.75), whereas adolescents had potentially decreased odds of severe complication (or 0.66, 95 % ci 0.391.14) following a severe pelvic fracture.table 6multivariate logistic regression analysis for isolated closed pelvic fractures with an associated ais 3, using predictors upon arrival to the emergency department with the outcomes death and severe complication (odds ratios and 95 % confidence intervals)model predictorsoutcomedeath, n = 2,487severe complication, n = 2,487children (< 13 years)1.90 (1.063.41)1.07 (0.651.75)adolescent (1317 years)0.40 (0.180.91)0.66 (0.391.14)adults (1854 years)referentreferentmale1.31 (0.951.80)not caucasian1.34 (0.902.01)hypovolemic shock9.93 (6.5215.1)6.56 (4.579.42)head injury1.70 (1.052.77)2.51 (1.723.66)iss 2519.3 (10.535.5)10.5 (7.2315.2)motor vehicle accident0.96 (0.621.50)0.98 (0.651.49)motorcycle accident1.27 (0.722.23)low - energy fall0.25 (0.061.12)0.96 (0.471.99)hosmer lemeshow0.290.14bold values signify 95 % confidence intervals that are completely greater than 1, indicating a significant positive associationiss injury severity scoresevere complication is defined as having one or more of the following during hospital course: pneumonia, bacteremia, deep vein thrombosis, pulmonary embolism, renal failure, acute respiratory distress syndrome, or deathhosmer lemeshow p - value given for model s goodness of fit instead of odds ratio and 95 % confidence interval multivariate logistic regression analysis for isolated closed pelvic fractures with an associated ais 3, using predictors upon arrival to the emergency department with the outcomes death and severe complication (odds ratios and 95 % confidence intervals) bold values signify 95 % confidence intervals that are completely greater than 1, indicating a significant positive association iss injury severity score severe complication is defined as having one or more of the following during hospital course: pneumonia, bacteremia, deep vein thrombosis, pulmonary embolism, renal failure, acute respiratory distress syndrome, or death hosmer lemeshow p - value given for model s goodness of fit instead of odds ratio and 95 % confidence interval in this model, an iss > 25, hypovolemic shock, and head injury yielded a substantial increase in odds of death (table 6). descriptive statistics of demographics, injury mechanism, injury severity, treatment, and complications are presented in table 1. after sustaining a pelvic fracture when separated by age group, adolescents appeared to have the best survival statistics (93.2 %), while children had the worst (89.8 %). adolescents had the lowest percentage of severe complication (11.4 %), children had a slightly higher percentage (13.0 %), and adults had the highest percentage (15.7 %).table 1frequency distribution of characteristics for the entire population of pelvic fractures, n = 37,784variableage grouptotal, n (%) children (< 13 years), n (%), n = 5,325adolescents (1317 years), n (%), n = 4,052adults (1854 years), n (%), n = 28,407gender male2,349 (44.8)1,888 (46.7)17,393 (61.3)21,630 (57.4) female2,897 (55.2)2,157 (53.3)10,983 (38.7)16,037 (42.6)race caucasian3,570 (72.3)2,745 (73.4)18,301 (69.9)24,616 (70.7) african american474 (9.6)341 (9.1)2,717 (10.4)3,532 (10.1) asian / pacific islander73 (1.5)54 (1.4)504 (1.9)631 (1.8) hispanic490 (9.9)394 (10.6)3002 (11.5)3,886 (11.2) native american31 (0.6)31 (0.9)201 (0.8)263 (0.8) other299 (6.1)173 (4.6)1,440 (5.5)1,912 (5.4)survival survived4,766 (89.8)3,753 (93.2)25,853 (91.5)34,372 (91.4) died542 (10.2)273 (6.8)2,404 (8.5)3,219 (8.6)severe complication yes693 (13.0)462 (11.4)4,465 (15.7)5,620 (14.9) no4,632 (87.0)3,590 (88.6)23,942 (84.3)32,164 (85.1)fracture type open183 (3.4)96 (2.4)1,316 (4.6)1,595 (4.2) closed5,142 (96.6)3,956 (97.6)27,078 (95.4)36,176 (95.8)fracture location ilium703 (13.7)622 (15.7)3,797 (13.9)5,122 (14.1) ischium162 (3.1)153 (3.9)1,151 (4.2)1,466 (4.0) pubis2,615 (42.0)1,632 (41.2)11,281 (41.4)15,078 (41.5) multiple330 (6.4)220 (5.5)1,799 (6.6)2,349 (6.5) unspecified673 (13.1)342 (8.6)2,328 (8.6)3,343 (9.2) other1,120 (21.7)996 (25.1)6,878 (25.3)8,994 (24.7)systolic blood pressure (mmhg) < 90878 (16.5)524 (12.9)4,615 (16.3)6,017 (15.9) 901392,869 (53.9)2,680 (66.2)16,704 (58.8)22,253 (58.9) 140 + 1,578 (29.6)848 (20.9)7,088 (24.9)9,514 (25.2)moi motor vehicle1,329 (24.9)2,224 (54.9)11,520 (40.6)15,073 (39.9) motorcycle119 (2.2)91 (2.3)2,674 (9.4)2,884 (7.6) pedestrian170 (3.2)102 (2.5)566 (1.9)838 (2.2) crush93 (1.8)31 (0.7)732 (2.6)856 (2.3) high fall592 (11.1)108 (2.6)2,680 (9.5)3,380 (9.0) low fall1,216 (22.8)63 (1.6)1,393 (4.9)2,672 (7.1) unknown1,806 (34.0)1,433 (35.4)8,842 (31.1)12,081 (31.9)ais of pelvis none3,323 (62.4)2,660 (65.6)19,331 (68.1)25,314 (67.0) 19 (0.2)11 (0.3)96 (0.3)116 (0.3) 21,313 (24.6)869 (21.5)5,153 (18.2)7,335 (19.4) 3610 (11.5)450 (11.1)3,129 (11.0)4,189 (11.1) 453 (1.0)45 (1.1)522 (1.8)620 (1.6) 517 (0.3)17 (0.4)176 (0.6)210 (0.6) 60 (0.0)0 (0.0)0 (0.0)0 (0.0)iss < 254,338 (81.5)2,816 (69.5)19,721 (69.4)26,875 (71.1) 25987 (18.5)1,236 (30.5)8,686 (30.6)10,909 (28.9)pneumonia yes102 (1.9)125 (3.1)1,232 (4.3)1,459 (3.9) no5,223 (98.1)3,927 (96.9)27,175 (95.7)36,325 (96.1)acute respiratory stress syndrome yes61 (1.1)55 (1.4)573 (2.0)689 (1.8) no5,264 (98.9)3,997 (98.6)27,834 (98.0)37,095 (98.2)deep vein thrombosis yes27 (0.5)21 (0.5)503 (1.8)551 (1.5) no5,298 (99.5)4,031 (99.5)27,904 (98.2)37,233 (98.5)bacteremia yes10 (0.2)26 (0.6)196 (0.7)232 (0.6) no5,315 (99.8)4,026 (99.4)28,211 (99.3)37,501 (99.4)renal failure yes32 (0.6)15 (0.4)225 (0.8)272 (0.7) no5,293 (99.4)4,037 (99.6)28,182 (99.2)37,512 (99.3)pulmonary embolism yes15 (0.3)5 (0.1)160 (0.6)180 (0.4) no5,310 (99.7)4,047 (99.9)28,247 (99.4)37,604 (99.6)icu stay < 1 week4,890 (91.8)3,545 (87.5)24,086 (84.8)32,521 (86.1) 1 week435 (8.2)507 (12.5)4,321 (15.2)5,263 (13.9)hospital stay < 1 week3,727 (70.0)2,406 (59.4)13,778 (48.5)19,911 (52.7) 1 week1,598 (30.0)1,646 (40.6)14,629 (51.5)17,873 (47.3)major procedure crif1 (0.02)2 (0.05)20 (0.07)23 (0.08) orif245 (4.62)395 (9.71)4,691 (16.47)5,331 (14.1) closed dislocation reduction7 (0.13)19 (0.47)117 (0.41)143 (0.39) open dislocation reduction12 (0.23)15 (0.37)127 (0.45)154 (0.43) no major procedure5,060 (95.0)3,621 (89.4)23,452 (82.6)32,133 (85.0)moi mechanism of injury, ais abbreviated injury scale, iss injury severity score, icu intensive care unit, crif closed reduction internal fixation, orif open reduction internal fixation frequency distribution of characteristics for the entire population of pelvic fractures, n = 37,784 moi mechanism of injury, ais abbreviated injury scale, iss injury severity score, icu intensive care unit, crif closed reduction internal fixation, orif open reduction internal fixation most of the pelvic fractures were closed (95.8 %). pubic rami fractures consistently had the highest prevalence across all age groups (41.5 %). the most common known mechanism of injury was motor vehicle accident (39.9 %), followed by high - energy fall (9.0 %) and motorcycle accident (7.6 %). most patients had an iss < 25 (71.1 %). when divided by age, more children had an iss < 25 (81.5 %) than adolescents (69.5 %) or adults (69.4 %). adolescents also had less reports of hypovolemic shock on arrival (systolic pressures below 90 mmhg) (12.9 %) than children (16.5 %) and adults (16.3 %) who had comparable numbers. for this assessment, open fractures and unknown mechanisms of injury were excluded, yielding a study group of 24,684. the of the bivariate analysis (reporting crude ors and 95 % cis) for this population are depicted in table 2. as compared to the rest of the population , children had a small increase in odds of death (or 1.27, 95 % ci 1.111.44), but a slight decrease in odds of severe complication (or 0.83, 95 % ci 0.750.93). adolescents had decreases in both odds of death (or 0.85, 95 % ci 0.721.00) and severe complication (or 0.78, 95 % ci 0.690.89). adults had a slight decrease in odds of death (or 0.92, 95 % ci 0.821.02), but a small increase in odds of severe complication (or 1.27, 95 % ci 1.161.39). individuals suffering from hypovolemic shock had substantially increased odds of death (or 5.89, 95 % ci 5.326.51) and severe complication (or 3.62, 95 % ci 3.333.93). sustaining a head injury also increased the odds of death (or 2.96, 95 % ci 2.593.39) and complication (or 3.23, 95 % ci 2.903.61). an iss 25 greatly increased the odds of death (or 11.1, 95 % ci 9.9212.5) and complication (or 9.19, 95 % ci 8.489.97). motor vehicle accidents caused the largest increase in odds of death (or 1.46, 95 % ci 1.321.62) and severe complication (or 1.53, 95 % ci 1.421.65).table 2bivariate analysis for the entire population of closed pelvic fractures comparing predictors upon arrival to the emergency department with the outcomes death and severe complication (odds ratios and 95 % confidence intervals), n = 24,684predictorsoutcomedeathsevere complicationmale1.24 (1.131.37)1.41 (1.311.52)not caucasian1.14 (1.041.26)1.00 (0.921.08)pediatric (< 18 years)1.09 (0.981.21)0.79 (0.720.86)children (< 13 years)1.27 (1.111.44)0.83 (0.750.93)adolescent (1317 years)0.85 (0.721.00)0.78 (0.690.89)adults (1854 years)0.92 (0.821.02)1.27 (1.161.39)hypovolemic shock5.89 (5.326.51)3.62 (3.333.93)head injury2.96 (2.593.39)3.23 (2.903.61)iss 2511.1 (9.9212.5)9.19 (8.489.97)motor vehicle accident1.46 (1.321.62)1.53 (1.421.65)motorcycle accident1.34 (1.161.54)1.34 (1.201.49)fall from height0.61 (0.520.72)0.61 (0.540.69)low - energy fall0.41 (0.330.51)0.36 (0.300.43)crush0.43 (0.290.63)0.66 (0.520.84)pedestrian versus auto1.40 (1.111.77)1.13 (0.931.38)iss injury severity scoresevere complication is defined as having one or more of the following during hospital course: pneumonia, bacteremia, deep vein thrombosis, pulmonary embolism, renal failure, acute respiratory distress syndrome, or death bivariate analysis for the entire population of closed pelvic fractures comparing predictors upon arrival to the emergency department with the outcomes death and severe complication (odds ratios and 95 % confidence intervals), n = 24,684 iss injury severity score severe complication is defined as having one or more of the following during hospital course: pneumonia, bacteremia, deep vein thrombosis, pulmonary embolism, renal failure, acute respiratory distress syndrome, or death variables that were found to be significant in the bivariate analysis were included in the final multiple logistic regression models for death and severe complication as outcomes. adjusted ors and 95 % cis are presented in table 3. when controlling for mechanism of injury and other prehospital conditions, children had more than twice the odds of death (or 2.29, 95 % ci 1.962.67) and increased odds of severe complication (or 1.36, 95 % ci 1.201.55) relative to the adult group after sustaining a pelvic fracture. adolescents, on the other hand, had a slight decrease in odds of death (or 0.89, 95 % ci 0.741.06) and lower odds of severe complication (or 0.70, 95 % ci 0.610.81) relative to the adult group after sustaining a pelvic fracture.table 3multivariate logistic regression analysis for the entire population of closed pelvic fractures using predictors upon arrival to the emergency department with the outcomes death and severe complication (odds ratios and 95 % confidence intervals)model predictorsoutcomedeath, n = 24,684severe complication, n = 22,846children (< 13 years)2.29 (1.962.67)1.36 (1.201.55)adolescents (1317 years)0.89 (0.741.06)0.70 (0.610.81)adults (1854 years)referentreferentmale1.19 (1.071.33)1.38 (1.261.50)not caucasian1.00 (0.901.12)hypovolemic shock4.52 (4.055.05)2.81 (2.563.08)head injury1.60 (1.371.86)1.70 (1.501.92)iss 259.15 (8.0710.4)7.41 (6.798.09)motor vehicle accident3.98 (0.3940.2)1.13 (0.921.41)motorcycle accident4.61 (0.4646.8)1.18 (0.921.51)fall from height3.27 (0.3233.1)0.80 (0.631.03)low - energy fall3.18 (0.3231.7)0.77 (0.581.02)pedestrian versus auto4.50 (0.4544.8)crush2.42 (0.2325.3)0.95 (0.691.34)hosmer lemeshow0.00690.17bold values signify 95 % confidence intervals that are completely greater than 1, indicating a significant positive associationiss injury severity scoresevere complication is defined as having one or more of the following during hospital course: pneumonia, bacteremia, deep vein thrombosis, pulmonary embolism, renal failure, acute respiratory distress syndrome, or deathhosmer lemeshow p - value given for the model s goodness of fit instead of odds ratio and 95 % confidence interval multivariate logistic regression analysis for the entire population of closed pelvic fractures using predictors upon arrival to the emergency department with the outcomes death and severe complication (odds ratios and 95 % confidence intervals) bold values signify 95 % confidence intervals that are completely greater than 1, indicating a significant positive association iss injury severity score severe complication is defined as having one or more of the following during hospital course: pneumonia, bacteremia, deep vein thrombosis, pulmonary embolism, renal failure, acute respiratory distress syndrome, or death hosmer lemeshow p - value given for the model s goodness of fit instead of odds ratio and 95 % confidence interval a subgroup analysis was performed for patients determined to have a severe, isolated, closed pelvic injury (ais 3). table 4 demonstrates the descriptive statistics for this subpopulation (n = 2,487). adolescents (n = 255) still appeared to have a lower incidence of mortality (3.1 %) and lower incidence of a severe complication (8.2 %) compared to adults (n = 1,886) and children (n = 346). adolescents contributed the most to motor vehicle accident (89.4 %), which was the most common mechanism of injury in the severe pelvic fracture group. similar to the whole population, the severe pelvic injury group demonstrated more children with an iss < 25 (82.1 %). for cases of hypovolemic shock on arrival, adolescents had the fewest reports of systolic pressures below 90 mmhg (6.7 %), while children (15.9 %) had the largest contribution. children had the lowest incidence of hospital stay and intensive care unit (icu) admission, while adolescents and adults were relatively comparable.table 4frequency distribution of characteristics for individuals with isolated, closed, severe pelvic fractures (ais3), n = 2,487variableage grouptotal, n (%) children (< 13 years), n (%), n = 346adolescents (1317 years), n (%), n = 255adults (1854 years), n (%), n = 1,886gender male127 (38.7)92 (36.2)1,109 (58.9)1,328 (53.8) female201 (61.3)162 (63.8)775 (41.1)1,138 (46.2)race caucasian235 (77.1)168 (74.3)1,208 (73.0)1,611 (73.7) african american31 (10.2)30 (13.3)170 (10.3)231 (10.6) asian / pacific islander4 (1.3)1 (0.4)24 (1.5)29 (1.3) hispanic29 (9.5)20 (8.9)200 (12.1)249 (11.4) native american1 (0.3)2 (0.9)7 (0.4)10 (0.5) other5 (1.6)5 (2.2)46 (2.7)56 (2.5)survival survived321 (92.8)247 (96.9)1,774 (94.1)2,342 (94.2) died25 (7.2)8 (3.1)112 (5.9)145 (5.8)severe complication yes32 (9.3)21 (8.2)209 (11.1)262 (10.5) no314 (90.8)234 (91.8)1,677 (88.9)2,225 (89.5)fracture location ilium41 (11.9)47 (18.4)228 (12.1)316 (12.7) ischium7 (2.0)7 (2.8)53 (2.8)67 (2.7) pubis222 (64.2)143 (56.1)1,059 (56.2)1,424 (57.3) multiple31 (9.0)30 (11.8)277 (14.7)338 (13.6) unspecified10 (2.9)4 (1.6)70 (3.7)84 (3.4) other35 (10.0)24 (9.3)199 (10.5)258 (10.3)systolic blood pressure (mmhg) < 9055 (15.9)17 (6.7)176 (9.3)248 (10.0) 90139173 (50.0)178 (69.8)1,197 (63.5)1,548 (62.2) 140 + 118 (34.1)60 (23.5)513 (27.2)691 (27.8)moi motor vehicle127 (36.7)228 (89.4)1,094 (58.0)1,449 (58.3) motorcycle17 (4.9)6 (2.4)196 (10.4)219 (8.8) pedestrian17 (4.9)5 (2.0)70 (3.7)92 (3.7) crush13 (3.8)5 (2.0)101 (5.4)119 (4.8) high fall52 (15.0)4 (1.5)248 (13.2)304 (12.2) low fall120 (34.7)7 (2.7)177 (9.3)304 (12.2)ais of pelvis 3321 (92.7)231 (90.6)1,606 (85.2)2,158 (86.8) 420 (5.8)17 (6.6)225 (11.9)262 (10.5) 55 (1.5)7 (2.8)55 (2.9)67 (2.7)iss < 25284 (82.1)169 (66.3)1,308 (69.4)1,761 (70.8) 2562 (17.9)86 (33.7)578 (30.6)726 (29.2)pneumonia yes3 (0.9)12 (4.7)53 (2.8)68 (2.7) no343 (99.1)243 (95.3)1,833 (97.2)2,419 (97.3)acute respiratory stress syndrome yes1 (0.3)1 (0.4)28 (1.5)30 (1.2) no345 (99.7)254 (99.6)1,858 (98.5)2,457 (98.8)deep vein thrombosis yes0 (0.0)1 (0.4)19 (1.0)20 (0.8) no346 (100.0)254 (99.6)1,867 (99.0)2,467 (99.2)bacteremia yes1 (0.3)1 (0.4)6 (0.3)8 (0.3) no345 (99.7)254 (99.6)1,880 (99.7)2,479 (99.7)renal failure yes2 (0.6)1 (0.4)7 (0.4)10 (0.4) no344 (99.4)254 (99.6)1,879 (99.6)2,477 (99.6)pulmonary embolism yes1 (0.3)0 (0.0)7 (0.4)8 (0.3) no345 (99.7)255 (100.0)1,879 (99.6)2,479 (99.7)icu stay < 1 week326 (94.2)224 (87.8)1,676 (88.9)2,226 (89.5) 1 week20 (5.8)31 (12.2)210 (11.1)261 (10.5)hospital stay < 1 week256 (74.0)151 (59.2)1,011 (53.6)1,418 (57.0) 1 week90 (26.0)104 (40.8)875 (46.4)1,069 (43.0)major procedure crif0 (0.0)0 (0.0)1 (0.1)1 (0.1) orif31 (9.0)45 (17.6)501 (26.5)577 closed dislocation reduction0 (0.0)2 (0.8)7 (0.4)9 (0.4) open dislocation reduction0 (0.0)0 (0.0)13 (0.7)13 (0.5) no major procedure315 (91.0)208 (81.6)1,364 (72.3)1,887 (75.9)moi mechanism of injury, ais abbreviated injury scale, iss injury severity score, icu intensive care unit, crif closed reduction internal fixation, orif open reduction internal fixation frequency distribution of characteristics for individuals with isolated, closed, severe pelvic fractures (ais3), n = 2,487 moi mechanism of injury, ais abbreviated injury scale, iss injury severity score, icu intensive care unit, crif closed reduction internal fixation, orif open reduction internal fixation similar to what was done with the whole population, bivariate analysis was also performed on the severe pelvic fracture subpopulation (table 5). as compared to the adults, children had higher odds of death but lower odds of severe complication. however, adolescents had a crude decrease in odds of death and severe complication after sustaining a pelvic fracture compared to the adults. individuals suffering from hypovolemic shock, head injury, and iss 25 had an increase in odds of death and severe complications. motor vehicle accidents caused the largest increase in odds of death (or 1.58, 95 % ci 1.102.26), while motorcycle accidents accounted for the largest increase in severe complication (or 1.58, 95 % ci 1.062.34).table 5bivariate analysis for isolated closed pelvic fractures with an associated ais 3, comparing predictors upon arrival to the emergency department with the outcomes death and severe complication (odds ratios and 95 % confidence intervals)predictorsoutcomedeath, n = 2,487severe complication, n = 2,487male1.11 (0.791.56)1.60 (1.222.08)not caucasian1.53 (1.092.15)1.26 (0.971.64)pediatric (< 18 years)0.92 (0.621.37)0.78 (0.571.06)children (< 13 years)1.31 (0.842.05)0.85 (0.571.25)adolescent (1317 years)0.50 (0.241.02)0.74 (0.471.18)adults (1854 years)1.09 (0.731.62)1.29 (0.941.77)hypovolemic shock13.0 (9.0618.6)7.35 (5.449.93)head injury4.41 (2.946.60)5.91 (4.288.16)iss 2527.5 (9.9212.5)14.9 (10.720.7)motor vehicle accident1.58 (1.102.26)1.32 (1.011.72)motorcycle accident1.62 (0.982.67)1.58 (1.062.34)fall from height0.63 (0.341.16)0.74 (0.481.13)low - energy fall0.095 (0.0230.38)0.35 (0.200.61)crush0.85 (0.371.97)0.95 (0.521.75)pedestrian versus auto1.33 (0.602.93)1.33 (0.602.93)bold values signify 95 % confidence intervals that are completely greater than 1, indicating a significant positive associationiss injury severity scoresevere complication is defined as having one or more of the following during hospital course: pneumonia, bacteremia, deep vein thrombosis, pulmonary embolism, renal failure, acute respiratory distress syndrome, or death bivariate analysis for isolated closed pelvic fractures with an associated ais 3, comparing predictors upon arrival to the emergency department with the outcomes death and severe complication (odds ratios and 95 % confidence intervals) bold values signify 95 % confidence intervals that are completely greater than 1, indicating a significant positive association iss injury severity score severe complication is defined as having one or more of the following during hospital course: pneumonia, bacteremia, deep vein thrombosis, pulmonary embolism, renal failure, acute respiratory distress syndrome, or death variables that were found to be significant in the bivariate analysis were included in the final multiple logistic regression models for death and severe complication as outcomes (table 6). when controlling for mechanism of injury and other prehospital conditions and using the adult group as a referent, children had nearly double the odds of death after sustaining a severe pelvic fracture, whereas adolescents had a large decrease in odds of death (or 0.40, 95 % ci 0.180.91). compared to adults, children had potentially increased odds of severe complication after sustaining a severe pelvic fracture (or 1.07, 95 % ci 0.651.75), whereas adolescents had potentially decreased odds of severe complication (or 0.66, 95 % ci 0.391.14) following a severe pelvic fracture.table 6multivariate logistic regression analysis for isolated closed pelvic fractures with an associated ais 3, using predictors upon arrival to the emergency department with the outcomes death and severe complication (odds ratios and 95 % confidence intervals)model predictorsoutcomedeath, n = 2,487severe complication, n = 2,487children (< 13 years)1.90 (1.063.41)1.07 (0.651.75)adolescent (1317 years)0.40 (0.180.91)0.66 (0.391.14)adults (1854 years)referentreferentmale1.31 (0.951.80)not caucasian1.34 (0.902.01)hypovolemic shock9.93 (6.5215.1)6.56 (4.579.42)head injury1.70 (1.052.77)2.51 (1.723.66)iss 2519.3 (10.535.5)10.5 (7.2315.2)motor vehicle accident0.96 (0.621.50)0.98 (0.651.49)motorcycle accident1.27 (0.722.23)low - energy fall0.25 (0.061.12)0.96 (0.471.99)hosmer lemeshow0.290.14bold values signify 95 % confidence intervals that are completely greater than 1, indicating a significant positive associationiss injury severity scoresevere complication is defined as having one or more of the following during hospital course: pneumonia, bacteremia, deep vein thrombosis, pulmonary embolism, renal failure, acute respiratory distress syndrome, or deathhosmer lemeshow p - value given for model s goodness of fit instead of odds ratio and 95 % confidence interval multivariate logistic regression analysis for isolated closed pelvic fractures with an associated ais 3, using predictors upon arrival to the emergency department with the outcomes death and severe complication (odds ratios and 95 % confidence intervals) bold values signify 95 % confidence intervals that are completely greater than 1, indicating a significant positive association iss injury severity score severe complication is defined as having one or more of the following during hospital course: pneumonia, bacteremia, deep vein thrombosis, pulmonary embolism, renal failure, acute respiratory distress syndrome, or death hosmer lemeshow p - value given for model s goodness of fit instead of odds ratio and 95 % confidence interval in this model, an iss > 25, hypovolemic shock, and head injury yielded a substantial increase in odds of death (table 6). in this study, we analyzed data gathered from the ntdb to characterize the risks for mortality and complications associated with pelvic fractures in adolescents as compared to younger children and adults. compared to adults, adolescents were shown to have decreased mortality and severe complication rates, while children have an increased mortality rate but decreased severe complication rate. in the subanalysis of severe pelvic fractures, the same pattern emerged, although the for complication rates were not statistically significant. mortality rates as low as 1.4 % to as high as 25 % have been reported. banerjee et al. reported a 16 % mortality rate in 44 patients related mainly to the associated injuries and not to the pelvic fractures themselves. another study reported 5 % overall mortality for 722 pediatric pelvic fractures versus 17 % among similar injuries in the adult population. comparable to previously reported studies, the current study reported 10.2 % survival for children and only 6.8 % for adolescents, compared to 8.5 % in adults with pelvic fractures. the pattern of lower survival in children and higher survival in adolescents compared to adults was also seen in the analysis of severe pelvic injuries (table 4). this study illustrates that simply comparing pediatric mortality to that of adults may be an inadequate evaluation due to the fact that adolescents and children sustaining pelvic injuries may have different outcomes. central nervous system head injury was cited as the most common cause of death in children with pelvic fractures in two recent studies. other causes of death in children with pelvic fractures, reported in these studies, include multiorgan failure and visceral injuries. in a study that examined the national pediatric trauma registry (nptr), hospital type (children s or general) and iss were the only significant parameters in the multivariate analysis. hemorrhage from a pelvis fracture - related vascular injury was reported as the cause of death in only 0.3 % of children, compared with 3.4 % of adults. multivariate analysis showed hypovolemic shock, head injury, and iss 25 to correlate with an increased mortality rate in a patient with any type of pelvic fracture (severe or not). the multivariate model for severe pelvic injuries also demonstrated that, despite their lower iss, children had a significantly higher risk of mortality (or 1.90, 95 % ci 1.063.41) and adolescents had a significantly lower risk of mortality (or 0.40, 95 % ci 0.180.91) as compared to adults. whereas many studies report the rate of associated injuries, very few studies report the rate of complications after pediatric pelvic fractures. according to a previous study of 120 children (age < 16 years) with pelvic fractures, complications included 13.3 % wound complications, 8.3 % urinary tract infections, 4.2 % septicemias, 1.7 % ards, and one case (0.8 %) of pulmonary embolus (pe). in the current study, there were 346 children (age < 13 years) with severe pelvic injuries, with a lower overall complication rate of 9.3 %. there was one case (0.3 %) of ards and one case of pe (0.3 %), two cases of renal failure (0.6 %), and no cases of septicemia. the 255 adolescents with severe pelvic injury had an even lower complications rate of 8.2 %, with one ards (0.4 %), one septicemia (0.4 %), one renal failure (0.4 %), one dvt, but no pe. urinary tract infection (uti) was not considered to be a significant complication in our study. however, in the severe pelvic injury group, there were three children (0.87 %), three adolescents (1.2 %), and 30 adults (1.59 %) with uti. the most common severe complication was pneumonia: three children (0.87 %), 12 adolescents (4.7 %), and 53 adults (2.8 %). previous studies have reported a high percentage of associated injuries in the pediatric pelvic fracture population. in one study surveying 166 cases , there were 38.6 % head traumas, 19.9 % significant chest traumas, and 19.3 % abdominal / visceral trauma. additionally, an incidence of 1127 % of associated abdominal injuries , 27 % associated chest injuries, and up to 50 % of associated head injuries have been previously reported. the proportion of associated injuries (head, thorax, and abdomen, respectively) in the current study was 7.5, 4.1, and 4.6 % for children; 18, 7.1, and 8.6 % for adolescents. the proportion of a depressed consciousness level in the population of severe pelvic fractures was 19.4 % in children and 31.3 % in adolescents (25 % if adolescents and children are grouped together). other pertinent findings in this study included a 57 times higher incidence of pelvic fractures in adults versus children and adolescents, a higher incidence of pelvic fractures in white non - hispanic males, the vast majority of the injury mechanism of pelvic fractures being blunt trauma from motor vehicle accidents, and a predominance of non - operative treatment for these fractures. these findings are largely in accordance with previous reports. the main strengths of the current study is that this is the largest cohort used for an investigation of this sort, and the national representation of academic and non - academic centers of various levels provide far - reaching generalizability. while the ntdb includes a very large sample of trauma centers from all over the united states, it is a convenience sample that consists solely of data submitted by participating hospitals, leading to a disproportionate number of larger hospitals with younger and more severely injured patients. the quality of data entered into the ntdb is solely dependent on the participants entering the data; however, the ntdb is continuously working to screen, clean, and standardize the quality of the data entered into the database. the larger population provided by the ntdb may be the reason for a higher mortality seen in our pediatric population, unlike the previous studies with smaller sample sizes. the ntdb also reported more pelvic fractures in children than in adolescents, which contributed to our pediatric population s higher mortality rate. we also used pelvis ais 3 to indicate a severe pelvic injury, while other studies have used a cutoff of ais 4. additionally, since the ntdb does not code separately for pelvic ais, we derived our pelvic ais score by using the lower extremity ais score and excluding all patients with other lower extremity injuries. assuming an associated lower extremity fracture rate of about 25 %, this may have influenced our descriptive statistics. however, this allowed us to compare similar injuries and better isolate the effect of age. the ntdb also does not provide blood gas values such as base deficit and lactate, and, therefore, our analysis was limited to the use of hypovolemic shock on arrival. this suggests that there could be interactions between variables that were not included in the model. these variables might not have been collected by the ntdb or may be latent factors that are not identified. such discrepancies may have altered our final parameter estimates by either inflating or deflating their values. finally, we chose the age group of 1317 years to represent adolescence. biological, as well as social and environmental, changes influence the beginning of adolescence, making the precise beginning age questionable. because of this, our age group was chosen based on previous publications. in , the of this study suggest that the adolescents with pelvic fractures behave as a separate group from the children and adult populations. these emphasize the need for further epidemiological research on disparate injury response between pediatric subpopulations defined by level of maturity. there should be further research aimed towards designating physiological criteria besides age by which to place pediatric patients in their respective subpopulations. this study highlights the fact that the mortality rate seen in a given pediatric population may not be an accurate measurement, as our lower mortality rate largely stems from the adolescents increased survival, while the children may actually have a higher mortality rate than the adults. meir marmor md was involved in the study design, data analysis, and writing.

pediatric pelvic fractures are associated with high - energy trauma and injury to other systems, leading to an increased incidence of complication and mortality. previous studies analyzed the pediatric population as a whole, including both children and adolescents. the purpose of this study was to examine whether adolescents with pelvic fracture have different complication and mortality rates compared to younger children and adults.methodsusing the national trauma data bank, 37,784 patients below the age of 55 years with pelvic fractures were identified and divided into children (age < 13 years), adolescents (age 1317 years), and adults (age > 17 years). descriptive statistics and bivariate and multivariate analyses were performed.children had an increased odds of death and complications (or 1.36, 95 % ci 1.201.55), whereas adolescents had a decrease in odds of death (or 0.89, 95 % ci 0.741.06) and complications (or 0.70, 95 % ci 0.610.81) compared to the adult population.adolescents with pelvic fractures exhibit a different physiologic response to the children and adult populations. this emphasizes the need to distinguish these subpopulations in future epidemiological research and treatment planning.

it naturally occurs in soil and water as well as the intestines, and it is responsible for nosocomial infections. there have been few reports about community acquired pneumonia of serratia. this report presents a 37-year - old man with hemoptysis, fever, and shortness of breath. the clinical and laboratory examinations revealed that the patient had pseudohemoptysis due to s. marcescens pneumonia, on an immunocompromised pattern, because of the coexistence of sarcoidosis (stage 1). the patient is healthy and asymptomatic after 1-year follow - up. to the best of the authors knowledge, this is the first reported case of a pseudohemoptysis in a patient with pulmonary sarcoidosis. serratia, is an opportunistic pathogen, mainly responsible for infecting the urinary system, respiratory tract, central nervous system, and bloodstream.1 infections of colonized individuals are associated with invasive devices, surgery, and immunocompromised states. serratia ubiquitously thrives in moist environments and frequently contaminates liquids and equipment. in the outpatient setting, it has been associated with community - acquired pneumonias, but only limited case reports exist, and usually are associated with some degree of immunocompromise. this paper reports a case where the poor living environment and coexistence of sarcoidosis were likely the predisposing factors that developed pneumonia from serratia. a 37-year - old man presented in the emergency department complaining of productive cough with blood - tinged sputum, intermittent fever, and shortness of breath for the last 20 days. his past medical history was remarkable only for arterial hypertension treated with atenolol. on examination his axillary temperature was 37.8c, blood pressure was 140/80 mm hg with a normal heart rate of 90 bpm, and oxygen saturation spo2: 94%. , the patient had a mildly elevated white blood cell count of 10,900 cells/l, with normal differential. the hemoglobin level was 10.6 g / dl, with a hematocrit of 32.5%; the platelet count was 455,000/l. a basic metabolic panel was notable for an elevated serum creatinine of 2.52 mg / dl, with urea of 57 mg / dl, and normal electrolytes. serum angiotensin converting enzyme was slightly elevated at 57 iu / ml (normal range 852 iu / ml). the acute renal failure was attributed to the serratia infection, since the patient did not previously have this disorder and serratia is known to induce renal failure.1 there was no lymphadenopathy observed in general examination, but chest x - radiography demonstrated bilateral hilar prominence, lower - right lung lobe opacification, and right hemidiaphragm elevation (figure 1). chest high - resolution computed tomography scan was performed according to the department s protocol for investigating the thorax. the scan revealed consolidation of the anterior segment of the right lower lobe, and bilateral paratracheal, subcarinal, and hilar lymphadenopathy (figure 2). pulmonary function testing after remission of the productive cough (2 days) showed a restrictive pattern with a forced vital capacity (fvc) of 49% predicted, a forced expiratory volume in one second (fev1) of 47% predicted, and an fev1/fvc ratio of 71%. the total lung capacity was 66% of predicted, and the residual volume was 79% of predicted. there was reduction of the diffusion lung capacity for carbon monoxide to 68% of predicted. serologic studies for human immunodef iciency, hepatitis b virus, and a tuberculin skin test were negative. urine antigen for streptococcus pneumoniae and legionella were given upon admission, but they were also negative. no evidence of inflammation, granulomas, malignancy, vasculitis, or alveolar wall necrosis were found on biopsy. cell / l, with 78% alveolar macrophages, 12% lymphocytes, and 10% neutrophils. balf flow cytometry showed that 69% of the lymphocytes were t - lymphocytes, 7% were b - lymphocytes, and the t4/t8 ratio was 1.9, while the blood t4/t8 ratio was 1.4. microbiology cultures of the balf grew serratia marcescens, which was sensitive to ciprofloxacin, tobramycin, and amikacin. the smear for acid - fast bacilli was negative, and cultures for mycobacteria remained negative after 6 weeks. the patient was treated with ciprofloxacin 500 mg twice daily for 3 weeks. after the third day of treatment, the cough improved, and the reddish appearance of the sputum completely resolved. on follow - up after 1 month, chest x - radiography presented complete remission on right lower lobe infiltrate, while the perihilar lymphadenopathy persisted. after 1-year follow - up, the patient remains asymptomatic, and on chest x - radiography the infiltrate has completely resolved, while enlargement of the perihilar lymph nodes persists (figures 3 and 4). this is a case of pseudohemoptysis due to pulmonary infection with s. marcescens and concomitant sarcoidosis unexpectedly found. s. marcescens is a gram - negative, rod - shaped bacterium in the enterobacteriaceae family, found in soil, water, plants, and human gut flora. at room temperature pseudohemoptysis, which is the appearance of bright reddish or pinkish colored sputum without the evidence of any red blood cells, has been well described in colonization or infection with serratia and is attributed to this pigment. in this patient, similarly to previously published cases, no red blood corpuscles were seen on direct microscopic examination of his sputum.2 lymph node hyperplasia occurs in serratia infections and may persist after treatment. this is due to the marked follicular, primary and secondary germinal center and medullar hyperplasia.3 in this case, the lymph node biopsy did not show any of these features but only nonnecrotizing granulomas without evidence of infection. the coexistence of sarcoidosis and opportunistic infection in the absence of any immunosuppressive therapy has previously been documented.4 an infection can be the presenting problem subsequently leading to the diagnosis of sarcoidosis, as in this case. in sarcoidosis immunopathogenesis, the immune system undergoes a reactivity change. primary features are a reduction in circulating cd4 + lymphocytes (t - helper cells), while their numbers in tissue increase, which is associated with a marked increase in tissue cytokine production, particularly interferon-, granulocyte - macrophage colony - stimulating factor, and interleukin-2.5,6 this t - helper 1 (th-1) cytokine profile recruits macrophages, eventually forming a granulomatous reaction.7 in contrast to these highly active tissue - based responses, the immunity related to the circulatory cells expression is depressed by cutaneous anergy and poor responses to antigen recall testing. in addition, the production of inhibitory cytokines by macrophages appears to interfere with the normal immune response. girard et al reported five cases and reviewed 65 additional cases of opportunistic infections associated with sarcoidosis.8 while the majority of these occurred in patients receiving corticosteroids and were accompanied by cd4 lymphocytopenia, they described opportunistic infections in four untreated individuals. observations have suggested a pathogenetic role of gram - negative infections in sarcoidosis by driving an interleukin-18 response as another feature of the th-1 pathway.9 this has been postulated for infections with hemophilus influenzae and moraxella catarrhalis.10 in summary, this report describes a rare case of an opportunistic infection in an outpatient setting. in this case, the sarcoidosis was the underlying condition that induced an infection by gram - negative bacilli. written informed consent was obtained from the patient upon discharge for publication of this case report and all accompanying images.

introductionserratia marcescens is a gram - negative bacillus which belongs to the family enterobacteriaceae. it is a facultative anaerobe and produces red pigment at room temperature. it naturally occurs in soil and water as well as the intestines, and it is responsible for nosocomial infections. there have been few reports about community acquired pneumonia of serratia.case presentationthis report presents a 37-year - old man with hemoptysis, fever, and shortness of breath. the clinical and laboratory examinations revealed that the patient had pseudohemoptysis due to s. marcescens pneumonia, on an immunocompromised pattern, because of the coexistence of sarcoidosis (stage 1).appropriate antibiotic therapy for serratia was administered, and the patient s symptoms regressed. the patient is healthy and asymptomatic after 1-year follow - up. to the best of the authors knowledge, this is the first reported case of a pseudohemoptysis in a patient with pulmonary sarcoidosis.

merkel cell carcinoma (mcc) is a highly aggressive skin malignancy mainly affecting elderly and immunosuppressed people. mcc is a rare tumour, but its incidence, especially in men, has risen during the last two decades in several countries. for instance, an annual increase of 8% between 1986 and 2001 has been reported for the united states, and in the netherlands, mcc incidence rates have doubled between 1993 and 2007. in scandinavia, however, incidence of mcc did not rise between 1995 and 2005. mcc owes its name to characteristics it shares with merkel cells (mcs), the neuroendocrine cells of the skin. interest in mcc has significantly increased during the last years due to the discovery of merkel cell polyomavirus (mcpyv) and its implication in mcc pathogenesis. studies on mcpyv and other molecular risk factors like mutations in the atoh1 gene have greatly advanced our understanding of mcc pathogenesis. the monoclonal integration of viral dna in a large proportion of mccs suggests that viral infection precedes tumourigenesis. furthermore, mcpyv t antigens are required for maintenance of virus - carrying mcc cell lines. binding of large t antigen to cell cycle regulators or of small t antigen to the translation regulator 4e - bp1 are potential mechanisms by which integrated mcpyv could transform cells. mcpyv may thus at least contribute to mcc formation, being the first molecular risk factor identified in mcc. additionally, mutations in several genes, including the tumour suppressor atoh1, encoding a transcription factor involved in merkel cell differentiation, and pik3ca, which codes for phosphatidylinositol 4,5-bisphosphate 3-kinase, catalytic subunit, alpha isoform, have been found in subsets of mcc. such mutations might either act in concert with mcpyv during tumourigenesis, or cause mcc formation in the absence of the virus. if it was known from which cell type mcc originates, one could develop more specific therapies which target these particular cells. in the present paper, we start with a brief discussion of cells of origin in cancer and subsequently elaborate on different cell types which have been hypothesized to be at the origin of mcc. last but not least, we briefly discuss how mcpyv might affect the potential mcc precursors. cells of origin in cancer have been defined as cells which acquire the first genetic hit or hits that culminate in the initiation of cancer. given the extended lifetime and self - renewal of physiological stem cells, these cells which assure homeostasis of rapidly self - renewing tissues therefore, it is not surprising that various studies have identified stem cells as cells of origin in cancer, for example, hematopoietic stem cells in chronic myeloid leukemia (cml) or crypt stem cells in intestinal cancer. in the skin, youssef et al. identified long - term resident progenitor cells of the interfollicular epidermis and the upper infundibulum as cells of origin in basal cell carcinoma (bcc), using clonal analysis in mice. these long - lived progenitor cells could, however, also be called epidermal stem cells according to sellheyer. it is noteworthy that this stem cell population is also the cell of origin of merkel cells in mice. furthermore, squamous cell carcinoma (scc) could be induced in hair follicle stem cells as well as in cells immediately exiting the bulge, but not in transit amplifying cells, which are more developmentally restricted. generally, it should be noted that the terms stem cells and progenitor cells are often used interchangeably, as a sharp distinction is not always easy. the second group of cells of origin in cancer are committed progenitor cells which differ from stem cells by their much more restricted differentiation potential. such cells play an important role in the acute phase of cml, but have also been identified as cells of origin in solid tumours. examples include medulloblastoma arising from granule neuron progenitors and breast cancer developing from luminal epithelial progenitors. third, even differentiated cells could give rise to cancer, as any cell which is able to proliferate could become a cell of origin in cancer, provided it acquires mutations which restore the ability to self - renew and prevent differentiation to a postmitotic state. for instance, a study in mice strongly suggests that malignant peripheral nerve sheath tumours arise from differentiated glia. moreover, differentiated endocrine cells in the pancreas appear to be a target for oncogenic transformation. however, it should be noted that in both cases, less differentiated progenitor cells can not be definitively excluded as cells of origin. before turning to the origin of mcc , it is important to keep in mind that the terms cell - of - origin in cancer and cancer stem cell are different. the cell - of - origin is a physiological cell which becomes tumourigenic due to genetic alterations. are a cell population within a tumour which constantly self - renews and is able to generate all types of cells present in this tumour, thus preserving the tumour. briefly, the cell - of - origin acquires tumourigenic properties, whereas the cancer stem cell sustains tumourigenic properties. regarding the cancer stem cell concept, early histological and ultrastructural analyses of the so - called trabecular carcinoma of the skin revealed similarities to merkel cells , leading to the currently used designation merkel cell carcinoma moreover, the reported morphological observations led to the that mcc may most probably originate from merkel cells (mcs). this traditional view of mcc origin (figure 1) was further corroborated by the discovery that mcc and mcs share a similar immunophenotype. shared features include presence of the merkel cell marker cytokeratin 20 (ck20) in mcc as well as biosynthesis of synaptophysin , ncam / cd56 , and numerous endocrine markers. although at first glance, these observations strongly support the hypothesis that mcc emerges from transformed mcs, there are quite a few data which question this view. for instance, the neural cell adhesion molecule l1 (cd171), a relative of ncam, is produced by mcc cells, but not by mcs. moreover, the arrangement of intermediate filaments, including ck20 and neurofilaments, differs between mcc and mcs: in mccs, whirl - or plaque - like aggregates are observed, whereas in mcs, the intermediate filament cytoskeleton is loosely and diffusely arranged. last but not least, the tyrosine kinase receptor c - kit, which has been detected in the majority of mccs, is mostly absent from human mcs. second, in a study on fetal and human skin, no proliferative merkel cells could be detected, strongly suggesting that human merkel cells are postmitotic. in line with these findings , more recent lineage tracing analyses revealed that in mice, adult merkel cell homeostasis is ensured by differentiation of epidermal progenitors, not through the proliferation of differentiated mcs. given that the possibility to restore proliferative potential is a prerequisite for a cell to become cell - of - origin in cancer, it is highly unlikely that postmitotic mcs would be the source of a malignancy. third, differences in tissue localization argue against mcs as cells - of - origin for mcc. whereas the majority of mcs is located in the basal layer of the epidermis, mccs are mostly found in the dermis and subcutis. however, it should be noted that a minority of 3.2% to 9.1% of reported cases are partly or even fully localized in the epidermis. finally, the observed heterogeneity in mcc rather favors less differentiated cells as cells of origin. in detail, mccs associated with diverse differentiation patterns have been described: squamous, squamous and sarcomatous, melanocytic, eccrine, leiomyosarcomatous, rhabdomyoblastic, and fibrosarcomatous differentiation. although there is a certain immunophenotypical diversity in mcs themselves, the multitude of differentiation patterns in mcc rather points to stem or early progenitor cells as cells - of - origin. it is, of course, conceivable that distinct stem or progenitor populations account for mccs with distinct differentiation patterns. in the following sections , we will therefore discuss stem cell populations in skin which might serve as cell of origin in mcc. as mentioned before, in mice, therefore, epidermal stem cells or long - lived epidermal progenitor cells appear to be good candidates for a mcc cell - of origin (figure 1). along this line, lemasson et al. as ck14 is a marker of the basal epidermal layer which comprises the epidermal stem cells, and as mcs in mice have ck14-positive progenitors , the authors argued that a malignant transformation of epidermal stem cells could underlie mcc, and that the transformed cells could then serve as cancer stem cells for mcc. in the same study, however, contradictory to the findings of lemasson et al., abbas and bhawan reported the absence of nestin in all 11 mcc cases investigated. it can not be excluded that these discrepancies were caused by different immunostaining protocols, as high variability of nestin immunoreactivity in skin due to procedural differences has been described. nevertheless, both studies showed the presence of ck19-positive cells in mcc. ck19 has been used as an epidermal stem cell marker, but is also present on mature merkel cells. this might be an argument against hair follicle bulge stem cells as cells of origin in mcc, as ck15 is found in these cells. summarizing, there are several hints pointing to an epidermal stem / progenitor cell origin of mcc, but no experimental proof so far. based on their histomorphological pattern as well as of immunohistochemical stainings, dermal neuroendocrine cells have been suggested as a potential source for mcc. this suggestion points to a second stem cell population present in skin, dermal stem cells (dscs) derived from the neural crest lineage (figure 1). interestingly, the transcription factor sox2, which is expressed by neural crest - derived stem cells from human skin, has been detected in mcc by immunohistochemistry. nine mcc samples were all sox2-positive, with more than 50% of cells exhibiting nuclear staining. on the other hand, sox2 is expressed by epidermal progenitors in the mouse tongue during their differentiation to sensory cells, demonstrating that it can not be regarded as an exclusive neural crest - derived stem cell marker. therefore, although mcc cells express several proteins abundant in neural cells, strong hints to a neural crest origin of mcc are currently missing. in addition to the neural crest - derived stem cells mentioned before, a further stem cell population has been isolated from murine and human dermis and termed skin - derived precursors (skps). these cells are able to differentiate into both neural and mesodermal cell types. in mice, facial skps are generated from the neural crest, whereas dorsal trunk skps derive from the somites (figure 1). although there are a lot of similarities between skps and the dscs mentioned before, both cell types differ in that skps lack the robust expression of the neurotrophin receptor p75 which is characteristic for dscs. given their dermal localization and their broad differentiation potential, skps should be regarded as a further potential cell of origin for mcc. if the mcpyv plays an important role in mcc genesis, as suggested by several recent studies (reviewed in), it should target the putative cells of origin (figure 1). it is noteworthy that jc polyomaviruses have been shown to preferentially infect stem cells or progenitor cells with a low degree of differentiation. if mcpyv exhibits a similar preference, this would argue for a stem rather than a merkel cell at the origin of mcc. along the same line, unpublished findings from our laboratory suggest that human merkel cells are normally devoid of mcpyv large t antigen (ltag). among 733 mcs detected by ck20 immunostaining in human skin areas prone to mcc development, none exhibited ltag immunopositivity in the adjacent section. although this does not prove the absence of mcpyv from differentiated mcs, it makes them less likely as a target for mcpyv infection. despite intense research on mcc during the last years, the cells of origin of this cutaneous malignancy remain elusive. merkel cells (mcs), originally the favourite candidate for such a role, appear less likely to give rise to mcc now, mainly because of mcc heterogeneity and the postmitotic character of mcs. stem cells located in the skin, most probably of the epidermal lineage, appear to be more probable cells of origin for mcc. however, as experimental evidence for a stem cell origin is missing, too, more direct approaches to tackle the origin - of - mcc - question are needed.

merkel cell carcinoma (mcc), a highly aggressive skin tumour with increasing incidence, is associated with the newly discovered merkel cell polyomavirus (mcpyv). studies on mcc and mcpyv as well as other risk factors have significantly increased our knowledge of mcc pathogenesis, but the cells of origin, which could be important targets in future therapies, are still unknown. merkel cells (mcs), the neuroendocrine cells of the skin, were believed to be at the origin of mcc due to their phenotypic similarities. however, for several reasons, for example, heterogeneous differentiation of mccs and postmitotic character of mcs, it is not very likely that mcc develops from differentiated mcs. skin stem cells, probably from the epidermal lineage, are more likely to be cells of origin in mcc. future studies will have to address these questions more directly in order to identify the physiological cells which are transformed to mcc cells.

cisplatin (cddp) has been the mainstay for the treatment of a broad spectrum of human malignancies since it was approved by the fda about 30 years ago. it has been used in the first - line treatment modalities of human malignancies, including testicular, ovarian , cervical, bladder, head and neck, and small cell lung cancers (sclcs). once patients develop resistance to platinum (pt) drugs, other effective treatment options become limited. it is well known that cddp acts on multiple cellular targets representing diverse signal transduction pathways. it is therefore conceivable that multiple mechanisms are involved in cddp resistance, including reduction of drug transport and increased dna adduct tolerance and repair. in this review , we focus on the role of glutathione (gsh) system in cddp resistance. the first mechanism involves the effects of gsh on the atp - binding cassette (abc) transporter - mediated cddp transport; the second mechanism involves the redox - regulating capacity of gsh in detoxifying cddp toxicity; the third mechanism involves regulation of the intracellular copper pool that affects cddp uptake. the abc transporters are a family of cytoplasmic membrane - spanned proteins that function as efflux pumps for eliminating antitumor agents, xenobiotics, and anionic lipophilic endogenous constituents. therefore, many of these transporters are also known as multidrug resistance proteins (mrps). gsh may serve as substrate for conjugation reaction with cddp prior to mrp - mediated transport. ishikawa and ali - osman first reported the formation of a pt(gs)2 conjugate in l1210 leukemia cells. these investigators also reported that elimination of pt(gs)2 across the membrane requires atp, suggesting the involvement of an energy - dependent transporter of gsh - conjugate (termed gs - x pump) in the elimination of pt(gs)2 complex. later, gene encoding mrp2, member abcc2 in the abc transporter family, was cloned; it was found that transfection of mrp2 into hek-293 cells conferred cddp resistance (10-fold) in the transfected cells. these demonstrated that mrp2 is the gs - x pump for the elimination of cddp. mrp2, like mrp1, can also transport gssg itself, an oxidized form of gsh, with relatively higher affinity than does gsh. thus, these abc transporters can be considered as regulators of intracellular gssg - gsh homeostasis and the associated redox maintenance (see below). many studies have demonstrated direct interactions between gsh and abc transporters , suggesting that gsh may induce conformational changes that facilitate mrp - mediated substrate transport. mrp2 is also known as a canalicular multispecific organic anion transporter (cmoat) because of its high level of expression in the hepatic canalicular compartment and because it mediates the transport of a broad spectrum of nonbile salt organic anions from the liver into bile. cmoat - deficient (tr-) wistar rats are mutated in the gene encoding mrp2, leading to defective hepatobiliary transport of a whole range of substrates, including bilirubin glucuronide. mrp2 mrna and protein levels can be markedly induced by treatments with metalloid salts including sodium arsenite and potassium antimonyl tartrate in primary rat and human hepatocytes. expression of mrp2 in primary rat hepatocytes is also induced by cddp. in one study, a single subcutaneous injection of cddp (5 mg / kg) into male sprague - dawley rats ed in > 10-fold induction of mrp2 in renal brush - border membranes within one day of treatment whereas nonsignificant induction of mrp2 levels was found in the livers. in normal rats, ~47% of the initial cddp dose the finding that increased expression of mrp2 in renal bbm upon injection of cddp suggests that this transporter may be involved in the excretion of cddp by the kidney. since levels of mrp2 are already high in the hepatocytes, this may explain why only marginal increases of mrp2 was seen in the livers of cddp - treated animals. moreover, a recent report showed that elevated mrp2 levels seemed to affect the efficacy of cddp - based chemotherapy in hepatocellular carcinoma hcc. while ishikawa and ali - osman initially reported that formation of pt(gs)2 complex reached a maximal level after 12 hrs in l1210 cells treated with 20 m cddp, corresponding to ~60% of the intracellular pt content, however, berners - price and kuchel studied the reaction of cddp with gsh inside intact red blood cells using h spin - echo nuclear magnetic resonance (nmr) and detected no formation of pt - gsh bonds within 4 hrs of incubation. recently, kasherman et al. studied the interactions of cddp with cell extracts prepared from ovarian cancer cells and found very little pt(gs)2 by the hsqc approach. since cddp can bind to many thiol - containing proteins and its primary cytotoxic target dna, once inside the cells, these cellular constituents will compete against gsh for cddp binding. these investigators found that the majority of glutathionated complexes were in fact in high molecular mass fraction (whereas the molecular mass of pt(gs)2 is 809 ). therefore, the significance of gsh - cddp binding as an important step for cddp elimination remains somewhat controversial. gsh is an abundant thiol - containing tri - peptide (glu - cys - gly), constituting 110 mm in mammalian cells. de novo biosynthesis of gsh is controlled by the rate - limiting enzyme, glutamate - cysteine ligase (gcl, also known as -glutamylcysteine synthetase, -gcs) which consists of a catalytic (heavy) (-gcsh) and a regulatory (light) subunit (-gcsl). -gcs carries out the initial ligating reaction of glutamine (glu) and cysteine (cys). production of gsh is accomplished by the subsequent reaction involving glycine (gly) by gsh synthetase (figure 1). gsh can be oxidized into gssg by gsh peroxidase using h2o2 as a substrate whereas gssg can be reduced back to gsh by gssg reductase using nadph as a cofactor. therefore, gsh - gssg system provides an important redox buffer in living organisms. as alluded to earlier, the transport activity of mrp2 (and other members of the mrp family) is regulated by gsh availability. since -gcs is the rate - limiting enzyme for the biosynthesis of gsh, coregulation of -gcs and mrps would facilitate the efflux activity (figure 1). indeed, we found that a number of cytotoxic agents can simultaneously induce the expression of both -gcsh and mrp, including cddp , carcinogens , and prooxidants. furthermore, enhanced expression of -gcsh / mrp1 was found in colorectal cancers, which are associated with inflammation - associated oxidative stress. these observations, taken together, strongly suggest that gsh/-gcs system is a molecular sensor of oxidative stress conditions. previous studies have reported that exposure of cultured cells to cddp led to the development of cddp resistance which was correlated with increased cellular gsh levels. moreover, gsh depletion by buthionine - sulfoximine (bso) has been associated with increased sensitivity to cddp. in many cases, when -gcs mrna contents were measured, elevated levels of -gcsh mrna were also correlated with cddp resistance. these studies have been widely taken as that intracellular gsh levels play an important role in regulating cddp resistance , perhaps through a gsh - mediated cytoprotective mechanism. however, molecular mechanisms as how gsh functions as a cytoprotector for cddp resistance have not been elucidated. one, these studies frequently used cddp - treated cells and the observations were mostly correlative in nature. whether elevated expression of gsh indeed is causally responsible for the cddp resistance needs to be critically evaluated. two, as alluded to above, induction of -gcs, and thus gsh, in cddp - treated cells may be an oxidative stress - induced phenomenon because -gcs / gsh is a sensor / regulator of reactive oxygen species (ross) imbalance, and its expression can be induced by a wide array of cytotoxic insults. in many cases, elevated expression of -gcs / gsh under cytotoxic insult three, also as alluded above, normal cells usually contain millimolar levels of gsh. stoichiometrically, these abundant amounts of thiol compound may already be sufficient to neutralize the cytotoxic effects of cddp which are usually in micromolar ranges. thus, increased gsh content found in most of cddp - treated cells may not necessarily have a major impact in the detoxification mechanism of cddp. as will be described below, we found that elevated expression gsh per se induces cellular sensitization to cddp treatment. both transcriptional regulation and posttranscriptional regulation have been reported for the upregulation of -gcsh by various cytotoxic assaults. transcriptional regulation is mediated by an antioxidant response element (are, 5-tgagtca) located at the promoter of the -gcsh allele, which interacts with the nf - e2-related transcription factor (nrf2). under unstressed conditions, majority of nrf2 is in the cytosol and bound to kelch - like ech - associated protein (keap1) which functions as a substrate adaptor for a cullin - dependent e2 ubiquitin ligase complex and targets nrf2 for ubiquitination and proteasomal degradation. because keap1 is a redox - sensitive e3 ligase, oxidative stress conditions induce keap1 sulfhydryl group modification and conformational changes, ing in nrf2 release from proteasomal degradation and allowing it to translocate to the nucleus. by heterodimerizing with the small maf protein as coactivator, together, they bind to the are and transactivate -gcsh expression. expression of mrp2 is also apparently regulated by nrf2 signal. livers from hepatocyte - specific gclc/-gcsh - knockout mice showed elevated expression of nrf2 and mrp2 due to elevated oxidative stress. we demonstrated that oxidative stress induces -gcsh mrna stabilization through the p38 map kinase pathways. under oxidative stress conditions, p38 map kinase activates mapkapk2, which promotes translocation of mrna - stabilizing factor hur from the nucleus to the cytoplasmic compartment, where it stabilizes -gcsh mrna by binding to the au - rich motif located at the 3 untranslated region. the accumulated -gcsh mrna produces elevated levels of gsh that feed back to suppress -gcsh mrna stabilization and the nrf2 signal - mediated -gcsh transcription as mentioned above. in 1998, while we were studying the coregulation of -gcsh and mrp1 by cddp, we found that transfection of recombinant dna encoding the -gcsh subunit alone was sufficient to enhance gsh levels in the transfected cells. surprisingly, we also found that these stable -gcsh - transfected cells exhibited hypersensitivity instead of resistance to the toxicity of cddp. hypersensitivity to the toxic effect of cddp was associated with enhanced uptake of cddp in these transfected cells. however, transporter for the uptake of cddp was not available at the time. cddp transporter was later identified as the high - affinity cu transporter (hctr1) and the mechanism of this hypersensitization was due to the upregulation of hctr1 in these transfected cells (see below). mutagenized yeast cells with a transpose library after selecting mutants that were able to grow in the presence of a toxic dose of cddp. one of the mutants was defective in the mac1 gene which encodes a copper concentration - dependent transcription factor for the expression of several genes involved in the uptake of iron and copper. these investigators subsequently determined that yctr1 is the target gene that could recapitulate the cddp - resistance phenotype observed in the mac1 mutant. likewise, murine embryonic fibroblasts derived from mctr1(/) animals exhibited reduced cddp accumulation as compared with their respective wild - type counterparts. the discovery that ctr1 is involved in pt drug transport underscores the important role of cu metabolism in the efficacy of pt drug chemotherapy. copper is an essential micronutrient for cell survival, yet cu overload is toxic. to meet their need for cu while avoiding toxicity, all living organisms from yeast to humans have developed an evolutionarily conserved system to regulate cu homeostasis. this system consists of a transporter (ctr1) for cu acquisition, chaperones (hah1, cox17, and ccs) for cu delivery to various intracellular compartments, and exporters (atp7a and atp7b) for cu elimination (figure 2). although cu entry can also be carried out by divalent metal transporter 1 (dmt1), which transports a broad range of divalent metal ions including cu(ii), the majority of cu acquisition is accomplished by ctr1, which transports cu(i). the belgrade rat, which has a mutation in dmt1, has no cu - deficient phenotype. extracellular cu exists in the oxidized form which is converted into cu(i) by membrane - bound cupric reductases, relevant to the yeast fre1 and fre2 reductases , for hctr1-mediated transport. expression of yeast ctr (yctr1 and yctr3) is transcriptionally upregulated under cu - deplete conditions and is downregulated under cu - replete conditions. during cu - depleted conditions, the transcription factor mac1p binds to the metal binding sequence located at the promoters of yctr1 and yctr3 and turns on the expression of these genes. under cu - replete conditions, mac1 dissociates from the promoters, ing in shut - down of the expression of yctr1 and yctr3. in the meantime, the transcription factor ace1 is activated to induce the expression of genes encoding cu - chelating proteins (cup1 and crs5) and the antioxidant superoxide dismutase (sod1) to protect cells from cu overload. both mac1 and ace1 contain zinc finger (zf) motifs that function as metallosensors. a transcriptional regulation mechanism is also involved in cu(i)-dependent regulation of the drosophila ctrb gene, a homologue of yctr1 and yctr3. in addition to transcriptional regulation, posttranslational regulation has been reported for yctr1 and yctr3 proteins in response to cu stress. available information in the literature regarding the mechanisms of regulation of human copper transporter (hctr1) is controversial. it has been reported that regulation of mammalian ctr1 is controlled at the posttranslational levels. elevated cu levels induce the trafficking of hctr1 from the plasma membrane to endosomal / lysosomal compartments where ctr1 is degraded. we investigated the regulation of hctr1 expression in response to cu concentration stress and found that levels of hctr1 mrna were decreased in cultured small cell lung cancer (sclc) cells treated with cuso4 but were increased in cells treated with the cu - depleting agent bathocuproine disulfonic acid. we further demonstrated that the zf transcription factor sp1 plays an important role in the transcriptional regulation of hctr1. these , taken together, indicate that expression of hctr1 is upregulated under copper deplete condition but is downregulated under copper replete conditions. the findings that hctr1 expression is regulated by intracellular cu bioavailability prompted us to investigate whether sensitization of gsh - overproducing cells in the -gcsh - transfected cells to cddp was due to reduced intracellular bioavailable cu contents, ing in upregulation of hctr1 expression. it has been well established that cu can form a cu - gsh complex by directly interacting with its internal cysteine - sh residue of gsh. however, unlike formation of pt(gs)2, formation of cu(gs) is almost a spontaneous reaction and requires no enzymatic involvement , ing in reduced intracellular bioavailability of cu levels. we found that the -gcsh - transfected cells indeed showed cu deficiency as evidenced by the reduced enzymatic activities of cu, zn superoxide dismutase (sod1) and mitochondrial cytochrome c oxidase (cco) as compared with those in the nontransfected cells. moreover, reduction of cu content in ceruloplasmin, a copper - containing ferroxidase that plays an important role in mammalian iron homeostasis, was also found in the transfected cells. levels of hctr1 expression in the three independently established -gcsh - transfected cells were 3- to 4-fold increases as compared with that in the parental cells. no substantial decreases in atp7a or atp7b levels were observed. moreover, increased hctr1 expression in these -gcsh - transfected cells was associated with enhanced cddp uptake. sensitization of -gcsh - transfected cells to cddp could be reversed when these cells were treated with gsh - depleting agent buthionine - sulfoximine (bso). this reversal was associated with the reduced expression of hctr1 due to the reinstated cu bioavailability in the transfected cells as analyzed by activities of the cu signature enzymes. taken together, our finding demonstrated that elevated levels of gsh per se can sensitize cddp toxicity. the elucidation that gsh functions as a cu chelator in upregulating its transporter hctr1 has important implications in cancer chemotherapy using platinum - based antitumor agents. we note that recent report has shown that another copper chelator, tetrathiomolybdate, can enhance cddp sensitivity in ovarian cancer animal model. the three major mechanisms that control cddp sensitivity by gsh described in the paper reflect the complexity of a small peptide that can regulate the efficacy of cddp toxicity. the significance of each of these mechanisms may depend upon various cell types and/or different cell physiologic conditions. as alluded to above, cddp can interact with many cellular targets and affect many signal transduction pathways to elicit its cytotoxicity. likewise, gsh is an important redox regulator and redox signaling can affect many important cellular processes. future investigations are needed to address the roles of gsh in the global effects of cddp sensitivity.

three mechanisms have been proposed for the role of glutathione (gsh) in regulating cisplatin (cddp) sensitivities that affects its ultimate cell - killing ability: (i) gsh may serve as a cofactor in facilitating multidrug resistance protein 2- (mrp2-) mediated cddp efflux in mammalian cells, since mrp2-transfected cells were shown to confer cddp resistance; (ii) gsh may serve as a redox - regulating cytoprotector based on the observations that many cddp - resistant cells overexpress gsh and -glutamylcysteine synthesis (-gcs), the rate - limiting enzyme for gsh biosynthesis; (iii) gsh may function as a copper (cu) chelator. elevated gsh expression depletes the cellular bioavailable cu pool, ing in upregulation of the high - affinity cu transporter (hctr1) which is also a cddp transporter. this has been demonstrated that overexpression of gsh by transfection with -gcs conferred sensitization to cddp toxicity. this review describes how these three models were developed and critically reviews their importance to overall cddp cytotoxicity in cancer cell treatments.

chd is a major cause of fetal loss and death in newborns less than one year of age all over the world. approximately, chd accounts for 28% of the major congenital anomalies (van der linde et al ., 2011). the generally accepted prevalence of chd was about 8 per 1000 live births, which poses a serious challenge to healthcare (bernier et al ., 2010). remarkable progresses have been achieved in chd diagnosis and cardiac surgery during the past decades, ing in an increased survival rate of neonates with chd (greutmann and tobler, 2012). however, more patients with chd have grown up who comprised of a special population: patients with grown - up congenital heart disease (guch) (khairy et al . it was reported that the prevalence of patients with guch was estimated to be 4 per 1000 adults . long - term medical care and related resource cost are needed for patients with guch, and rapidly increase healthcare burden . thus, it is very important to characterize the etiology of congenital heart disease, which has not been well understood yet . washington infant study have indicated that the cause of chd was multifactorial, and both genetic and environmental factors may play important roles in the development of chd ( richards and garg, 2010 ; shieh et al ., importantly, due to the advances in molecular techniques, accumulating evidences have suggested that genetic factors were dominant ( bruneau, 2008). it was known that a large proportion of chds were characterized with aneuploidy or abnormal chromosomal number (blue et al . , 2012 ; pierpont et al ., 2007). about 50% of children who were born with trisomy 21 have atrial and ventricular septal defects or atrioventricular canal lesion. with completion of the human genome project it has been reported that the mutations in single genes including tbx5, jag1, nkx2.5 and gata4 have been associated with the development of chd (basson et al ., 1997 ; the association between folic acid metabolism and the development of chd has been explored recently . maternal supplement of folic acid has been proved to reduce the incidence of chd as well as other congenital heart disease ( van beynum et al ., 2010). single nucleotide polymorphisms of many genes involved in the folate pathway have been identified to affect the function of the genes or folic acid metabolism and thus increase the risk of chd (locke et al ., 2010 ; shaw et al ., 2009). the flavin adenine dinucleotide - dependent enzyme 5,10-methylenetetrahydrofolate reductase (mthfr) catalyzes the reduction of methylenetetrahydrofolate to 5-methyltetrahydrofolate, which is required for the remethylation of homocysteine to methionine (ueland et al ., 2001) hyperhomocysteinemia was believed to be a high risk for the development of heart defects (verkleij - hagoort et al . , 2006, 2007). elevating the level of 5-methyltetrahydrofolate, a major circulating folic acid, prevented chd by reducing maternal homocysteine plasma level (lamers et al ., 2004). therefore, the polymorphisms of mthfr may be closely related to the risk of chd. it was reported that two mthfr snps including mthfr c677 t (p.ala222val, i d : rs1801133) and mthfr a1298c (p.glu429ala, rs1801131) were potentially associated with chd (van driel et al ., 2008). the amino acid transition in mthfr c677 t (ala - val) has ed in a thermolabile protein associated with reduced enzyme activity in vivo, which may increase plasma homocysteine level (huhta and hernandez - robles, 2005). the mthfr a1298 c has also been reported to moderately reduce mthfr activity in vivo (weisberg et al ., 1998). to date, a large number of studies regarding the associations between mthfr gene polymorphisms and risk of chd have been published. herein, we performed a meta - analysis of all published studies until january 2013 to investigate the association between the two snps (mthfr 677ct and mthfr 1298ac) and chd patients and their mothers. the pubmed, embase, web of knowledge and cnki (china national knowledge infrastructure) database searches were performed to identify all the eligible papers. the search terms were used as the following: (mthfr or methylenetetrahydrofolate reductase or folic acid) and (variant or polymorphism or snp) and (congenital heart disease or heart defect or chd or congenital anomalies). moreover, potentially relevant studies were evaluated by reviewing the titles and abstracts, and studies matching the criteria were carefully retrieved. if more than one study was published using the same data, only the study with a larger population was included. the eligible studies should meet the following inclusion criteria: investigation of association between the mthfr polymorphisms (including c677 t or a1298c or both) and congenital heart disease; a case control study; providing sufficient data on genotype frequencies of the mthfr c677 t and/or a1298c polymorphisms and sufficient data for calculation of an odd ratio (or) with 95% confidence interval (ci). the exclusion criteria were as follows: reviews, case report, editorial or comment; a duplicated study; studies providing insufficient data or data in poor quality; and studies without control. based on the inclusion and exclusion criteria, data extraction from each study was performed by two authors (wang, hou) independently to ensure that the data extraction were accurate. the following information was extracted from each study: name of the first author; year of publication; country of origin; ethnicity of the study population; source of controls (population based or hospital based); sample size of case and controls; types of congenital heart disease; genotype distributions in cases and controls; and whether population involved in the study was in hardy weinberg equilibrium (hwe). meta - analysis was performed to evaluate the association between mthfr polymorphisms and risk of developing chd. firstly, crude ors with 95% cis were calculated to assess the strength of the correlation between the mthfr c677t / a1298c polymorphisms (including maternal and fetal) and risk of chd. pooled ors and 95% cis were calculated for the multiplicative, co - dominant, dominant, and recessive genetic models respectively. the significances of pooled ors were analyzed by z tests, and the criteria for statistically significant were p < 0.05. a q test was conducted to determine the possible heterogeneity, and p < 0.10 or i > 50% indicated an obvious heterogeneity. pooled ors (95% ci) were calculated by random effects model (dersimonian laird method) or fix effects model (mantel haenszel method). subgroup analysis was performed by ethnicity, types of chd, source of controls and sample size (n < 100 vs. n > 100). sensitivity analysis were performed to evaluate the stability of the by removing one case control study each time to assess the influence of the individual data on pooled ors. moreover, the egger quantitative tests were also performed, and p < 0.05 was considered statistically significant. to obtain reliable data, two authors (wang, hou) have performed the statistical analysis independently by using the same data and the programs. data analyses were performed using stata version 12 (stata corporation, college station, texas, usa). the pubmed, embase, web of knowledge and cnki (china national knowledge infrastructure) database searches were performed to identify all the eligible papers. the search terms were used as the following: (mthfr or methylenetetrahydrofolate reductase or folic acid) and (variant or polymorphism or snp) and (congenital heart disease or heart defect or chd or congenital anomalies). moreover, potentially relevant studies were evaluated by reviewing the titles and abstracts, and studies matching the criteria were carefully retrieved. if more than one study was published using the same data, only the study with a larger population was included. the eligible studies should meet the following inclusion criteria: investigation of association between the mthfr polymorphisms (including c677 t or a1298c or both) and congenital heart disease; a case control study; providing sufficient data on genotype frequencies of the mthfr c677 t and/or a1298c polymorphisms and sufficient data for calculation of an odd ratio (or) with 95% confidence interval (ci). the exclusion criteria were as follows: reviews, case report, editorial or comment; a duplicated study; studies providing insufficient data or data in poor quality; and studies without control. based on the inclusion and exclusion criteria, data extraction from each study was performed by two authors (wang, hou) independently to ensure that the data extraction were accurate. the following information was extracted from each study: name of the first author; year of publication; country of origin; ethnicity of the study population; source of controls (population based or hospital based); sample size of case and controls; types of congenital heart disease; genotype distributions in cases and controls; and whether population involved in the study was in hardy weinberg equilibrium (hwe). meta - analysis was performed to evaluate the association between mthfr polymorphisms and risk of developing chd. firstly, crude ors with 95% cis were calculated to assess the strength of the correlation between the mthfr c677t / a1298c polymorphisms (including maternal and fetal) and risk of chd. pooled ors and 95% cis were calculated for the multiplicative, co - dominant, dominant, and recessive genetic models respectively. the significances of pooled ors were analyzed by z tests, and the criteria for statistically significant were p < 0.05. a q test was conducted to determine the possible heterogeneity, and p < 0.10 or i > 50% indicated an obvious heterogeneity. pooled ors (95% ci) were calculated by random effects model (dersimonian laird method) or fix effects model (mantel haenszel method). subgroup analysis was performed by ethnicity, types of chd, source of controls and sample size (n < 100 vs. n > 100). sensitivity analysis were performed to evaluate the stability of the by removing one case control study each time to assess the influence of the individual data on pooled ors. moreover, the egger quantitative tests were also performed, and p < 0.05 was considered statistically significant. to obtain reliable data, two authors (wang, hou) data analyses were performed using stata version 12 (stata corporation, college station, texas, usa). totally, we have identified 288 potentially relevant studies by employing the search strategy described above. based on obvious irrelevance to mthfr and chd in titles, 248 papers from the 288 potentially relevant papers were excluded. after reading the abstracts of the remaining 40 studies, 7 studies were further excluded, as 6 studies were reviews and one study was a duplicated study. to further polish target studies, of these, 9 studies were excluded, due to insufficient data, data with poor quality or papers without control. after careful screening, 24 eligible studies were finally included in this meta - analysis (balderrabano - saucedo et al ., 2013 ; bozovic et al ., 2011 ; galdieri et al ., 2007 ; garcia - fragoso et al ., 2010 ; gong et al . , 2009, 2012 ; hobbs et al ., 2010 ; junker et al ., 2001 , 2005, 2009a, 2009b ; liu et al ., 2005 ; peng et al ., 2009 ; sanchez - urbina et al ., 2012 ; shaw et al ., 2005 ; storti et al . van driel et al ., 2008 ; wang, 2006 ; wintner et al ., 2007 ; xu et al ., 2010 ; yan and li, 2003 ; zhu et al ., 2006). the search strategy and inclusion / exclusion of studies were shown in a flow chart (fig . 1). among these studies, fourteen studies investigated the maternal mthfr c677 t polymorphism with 1745 cases and 2044 controls and nineteen studies investigated the fetal mthfr c677 t polymorphism with 2697 cases and 3434 controls. in addition, there were 4 studies investigating maternal mthfr a1298c polymorphism with 432 cases and 532 controls and 5 studies investigating fetal mthfr a1298c polymorphism with 945 cases and 1074 controls. 9 studies included both maternal and fetal mthfr polymorphisms and 5 studies included both mthfr c677 t and a1298c polymorphisms. moreover, 11 studies were performed in caucasian and 12 studies were performed in asian. the genotype and allele distributions of maternal c677 t and a1298c polymorphisms in all the studies included were shown in table 2. for the fetal polymorphisms, the genotype and allele frequencies of c677 t and a1298c were shown in table 3. for the maternal mthfr c677 t polymorphism, the indicated no statistically significant association between the polymorphism and the susceptibility to chd in all genetic models except for recessive model and co - dominant model (t vs. c : or = 1.103, 95% ci 0.9991.218 ; tt vs. cc : or = 1.254, 95% ci 1.0121.553 ; tt + ct vs. cc : 1.105, 95% ci 0.9581.275 ; tt vs. tc + cc : or = 1.235, 95% ci 1.0241.489) (table 4 fig . 2). in the subgroup analysis by ethnicity, no significant association was observed in asian population in all genetic models except for recessive model (t vs. c : or = 1.204, 95% ci 0.9731.490 ; tt vs. cc : or = 1.454, 95% ci 0.9212.295 ; tt + ct vs. cc : or = 1.170, 95% ci 0.8021.707 ; tt vs. tc + cc : or = 1.431, 95% ci 1.0112.024) (table 4). no association was detected in caucasians in all genetic models. in the stratified analysis by types of chd , there was a significant association between c667 t and asd / pda, however, the were not reliable because only one study was performed in asd / pda patients (table 4). in the subgroup of source of control, association was only observed in the recessive model of hospital based control subgroup (tt vs. tc + cc : or = 1.550, 95% ci 1.1062.170) (table 4). in the sample size subgroup analysis , there was no significant association between chd and maternal c677 t in all genetic models of large sample studies. however, we have observed a significant association in co - dominant model and recessive model with small sample studies (table 4). for the fetal mthfr c667 t polymorphism, the overall suggested a significant association of polymorphism with chd susceptibility (t vs. c : or = 1.271, 95% ci 1.1781.372 ; tt vs. cc : or = 1.610, 95% ci 1.3741.885 ; tt + ct vs. cc : or = 1.258, 95% ci 1.1201.414 ; tt vs. tc + cc : or = 1.565, 95% ci 1.3701.788) (table 5, fig . fetal mthfr c677 t was associated with chd in asian populations for all genetic models, however, no significant association was found in caucasian ( table 5). in the stratified analysis by types of chd , significant associations were detected between fetal mthfr c677 t and all types of chd for all genetic models (table 5). similar significant association was also observed in cd and asd / pda, however, the positive in cd was not reliable because it was derived from one study. interestingly, a significant association was observed in hospital based control subgroup rather than in population based control subgroup. by considering sample size, significant were also found in all genetic models in both small and large sample subgroups (table 5). for mthfr a1298c polymorphism, the showed no significant association between this polymorphism and chd risk in either maternal or fetal groups (maternal : t vs. a : or = 1.043, 95% ci 0.8551.271 ; cc vs. aa or = 1.109, 95% ci 0.6921.775 ; cc + ac vs. aa : or = 1.108, 95% ci 0.8561.435 ; cc vs. ac + aa : or = 0.735, 95% ci 0.4671.157 ; fetal : c vs. a or = 0.938, 95% ci 0.8121.083 ; cc vs. aa : or = 1.058, 95% ci 0.7191.558 ; cc + ac vs. aa : or = 0.871, 95% ci 0.7281.042 ; cc vs. ac + aa : or = 1.184, 95% ci 0.8151.721). in the subgroup analysis of either maternal or fetal polymorphisms, there was no statistically significant association in each subgroup by ethnicity, types of chd, source of controls and sample size under all genetic models (table 6). as shown in table 4, heterogeneity between studies was significant (p < 0.10) under additive, and recessive genetic models for maternal mthfr c677 t. moreover, evidence for heterogeneity between studies was also found in all genetic models for fetal mthfr c677 t. for the mthfr a1298c polymorphism no evidence for heterogeneity between studies was detected for maternal mthfr c677 t in the co - dominant and dominant models, for maternal and fetal mthfr a1298c under all genetic models except for maternal recessive model. in the sensitivity analysis, the overall association between the maternal mthfr c677 t genotype and chd was not substantially changed by excluding one study at a time (data not shown). similar were also found in fetal mthfr c677 t, maternal and fetal mthfr a1298c. except for dominant model of fetal mthfr c677 t and co - dominant model of maternal a1298c polymorphisms, no publication bias could be detected by employing egger's test for studies on maternal mthfr c677 t polymorphism (t vs. c : p = 0.647 ; tt vs. cc : p = 0.324 ; tt + ct vs. cc p = 0.533 ; tt vs. tc + cc : p = 0.269); fetal mthfr c677 t polymorphism (t vs. c : p = 0.077 ; tt vs. cc : p = 0.110 ; tt vs. tc + cc : p = 0.057); maternal mthfr a1298c polymorphism (c vs. a : p = 0.882 ; cc + ac vs. aa : p = 0.330 ; cc vs. ac + aa : p = 0.107); and fetal mthfr a1298c polymorphism (c vs. a : p = 0.493 ; cc vs. aa : p = 0.576 ; cc + ac vs. aa : p = 0.576 ; cc vs. ac + aa : p = 0.576). the of egger's test suggested publication bias in dominant model of fetal mthfr c677 t polymorphism (tt + ct vs. cc : p = 0.006) and co - dominant model of maternal a1298c polymorphism (cc vs. aa : p = 0.049). the begg's tests of corresponding genetic models in forest plots were shown in figs. 2 and 3. totally, we have identified 288 potentially relevant studies by employing the search strategy described above. based on obvious irrelevance to mthfr and chd in titles, 248 papers from the 288 potentially relevant papers were excluded. after reading the abstracts of the remaining 40 studies, 7 studies were further excluded, as 6 studies were reviews and one study was a duplicated study. to further polish target studies, of these, 9 studies were excluded, due to insufficient data, data with poor quality or papers without control. after careful screening, 24 eligible studies were finally included in this meta - analysis (balderrabano - saucedo et al ., 2013 ; bozovic et al ., 2011 ; galdieri et al ., 2007 ; garcia - fragoso et al ., 2010 ; gong et al . , 2009, 2012 ; hobbs et al ., 2010 ; junker et al ., 2001 , 2005, 2009a, 2009b ; liu et al ., 2005 ; peng et al ., 2009 ; sanchez - urbina et al ., 2012 ; shaw et al ., 2005 ; storti et al . van driel et al ., 2008 ; wang, 2006 ; wintner et al ., 2007 ; xu et al ., 2010 ; yan and li, 2003 ; zhu et al ., 2006). the search strategy and inclusion / exclusion of studies were shown in a flow chart (fig . 1). among these studies, fourteen studies investigated the maternal mthfr c677 t polymorphism with 1745 cases and 2044 controls and nineteen studies investigated the fetal mthfr c677 t polymorphism with 2697 cases and 3434 controls. in addition, there were 4 studies investigating maternal mthfr a1298c polymorphism with 432 cases and 532 controls and 5 studies investigating fetal mthfr a1298c polymorphism with 945 cases and 1074 controls. 9 studies included both maternal and fetal mthfr polymorphisms and 5 studies included both mthfr c677 t and a1298c polymorphisms. moreover, 11 studies were performed in caucasian and 12 studies were performed in asian. the genotype and allele distributions of maternal c677 t and a1298c polymorphisms in all the studies included were shown in table 2. for the fetal polymorphisms, the genotype and allele frequencies of c677 t and a1298c were shown in table 3. for the maternal mthfr c677 t polymorphism, the indicated no statistically significant association between the polymorphism and the susceptibility to chd in all genetic models except for recessive model and co - dominant model (t vs. c : or = 1.103, 95% ci 0.9991.218 ; tt vs. cc : or = 1.254, 95% ci 1.0121.553 ; tt + ct vs. cc : 1.105, 95% ci 0.9581.275 ; tt vs. tc + cc : or = 1.235, 95% ci 1.0241.489) (table 4 fig . 2). in the subgroup analysis by ethnicity, no significant association was observed in asian population in all genetic models except for recessive model (t vs. c : or = 1.204, 95% ci 0.9731.490 ; tt vs. cc : or = 1.454, 95% ci 0.9212.295 ; tt + ct vs. cc : or = 1.170, 95% ci 0.8021.707 ; tt vs. tc + cc : or = 1.431, 95% ci 1.0112.024) (table 4). no association was detected in caucasians in all genetic models. in the stratified analysis by types of chd , there was a significant association between c667 t and asd / pda, however, the were not reliable because only one study was performed in asd / pda patients (table 4). in the subgroup of source of control, association was only observed in the recessive model of hospital based control subgroup (tt vs. tc + cc : or = 1.550, 95% ci 1.1062.170) (table 4). in the sample size subgroup analysis , there was no significant association between chd and maternal c677 t in all genetic models of large sample studies. however, we have observed a significant association in co - dominant model and recessive model with small sample studies (table 4). for the fetal mthfr c667 t polymorphism, the overall suggested a significant association of polymorphism with chd susceptibility (t vs. c : or = 1.271, 95% ci 1.1781.372 ; tt vs. cc : or = 1.610, 95% ci 1.3741.885 ; tt + ct vs. cc : or = 1.258, 95% ci 1.1201.414 ; tt vs. tc + cc : or = 1.565, 95% ci 1.3701.788) (table 5, fig . fetal mthfr c677 t was associated with chd in asian populations for all genetic models, however, no significant association was found in caucasian ( table 5). in the stratified analysis by types of chd , significant associations were detected between fetal mthfr c677 t and all types of chd for all genetic models (table 5). similar significant association was also observed in cd and asd / pda, however, the positive in cd was not reliable because it was derived from one study. interestingly, a significant association was observed in hospital based control subgroup rather than in population based control subgroup. by considering sample size, significant were also found in all genetic models in both small and large sample subgroups (table 5). for mthfr a1298c polymorphism, the showed no significant association between this polymorphism and chd risk in either maternal or fetal groups (maternal : t vs. a : or = 1.043, 95% ci 0.8551.271 ; cc vs. aa or = 1.109, 95% ci 0.6921.775 ; cc + ac vs. aa : or = 1.108, 95% ci 0.8561.435 ; cc vs. ac + aa : or = 0.735, 95% ci 0.4671.157 ; fetal : c vs. a or = 0.938, 95% ci 0.8121.083 ; cc vs. aa : or = 1.058, 95% ci 0.7191.558 ; cc + ac vs. aa : or = 0.871, 95% ci 0.7281.042 ; cc vs. ac + aa : or = 1.184, 95% ci 0.8151.721). in the subgroup analysis of either maternal or fetal polymorphisms , there was no statistically significant association in each subgroup by ethnicity, types of chd, source of controls and sample size under all genetic models (table 6). as shown in table 4, heterogeneity between studies was significant (p < 0.10) under additive, and recessive genetic models for maternal mthfr c677 t. moreover, evidence for heterogeneity between studies was also found in all genetic models for fetal mthfr c677 t. for the mthfr a1298c polymorphism, significant heterogeneity was only found in recessive model of maternal polymorphism. no evidence for heterogeneity between studies was detected for maternal mthfr c677 t in the co - dominant and dominant models, for maternal and fetal mthfr a1298c under all genetic models except for maternal recessive model. in the sensitivity analysis, the overall association between the maternal mthfr c677 t genotype and chd was not substantially changed by excluding one study at a time (data not shown). similar were also found in fetal mthfr c677 t, maternal and fetal mthfr a1298c. except for dominant model of fetal mthfr c677 t and co - dominant model of maternal a1298c polymorphisms, no publication bias could be detected by employing egger's test for studies on maternal mthfr c677 t polymorphism (t vs. c : p = 0.647 ; tt vs. cc : p = 0.324 ; tt + ct vs. cc p = 0.533 ; tt vs. tc + cc : p = 0.269); fetal mthfr c677 t polymorphism (t vs. c : p = 0.077 ; tt vs. cc : p = 0.110 ; tt vs. tc + cc : p = 0.057); maternal mthfr a1298c polymorphism (c vs. a : p = 0.882 ; cc + ac vs. aa : p = 0.330 ; cc vs. ac + aa : p = 0.107); and fetal mthfr a1298c polymorphism (c vs. a : p = 0.493 ; cc vs. aa : p = 0.576 ; cc + ac vs. aa : p = 0.576 ; cc vs. ac + aa : p = 0.576). the of egger's test suggested publication bias in dominant model of fetal mthfr c677 t polymorphism (tt + ct vs. cc : p = 0.006) and co - dominant model of maternal a1298c polymorphism (cc vs. aa : p = 0.049). the begg's tests of corresponding genetic models in forest plots were shown in figs. 2 and 3. to date, it is known that genetic and environmental risks may be the causes of congenital heart diseases. importantly, numerous studies have suggested the role of folic acid metabolism in the chd development (ueland et al ., 2001). mthfr is a key enzyme in folic acid conversion process, and its activity may be related with a variety of diseases including chd (li et al ., 2013 ; long et al ., 2012). it was reported that the c677 t mutation of mthfr could render the enzyme thermolabile with approximately 50% reduced activity and increased plasma homocysteine concentrations (huhta and hernandez - robles, 2005). therefore, the variants of the mthfr gene may modulate the activity of mthfr and may be an important determinant of chd development. several studies have reported the potential association between mthfr polymorphisms (c677 t and a1298c) and chd, however, the were not consistent (balderrabano - saucedo et al ., 2013 ; two studies have performed meta - analysis in association between mthfr c677 t polymorphism and chd two years ago ( nie et al ., 2011 ; yin et al ., 2012). however, the mthfr a1298c polymorphism was not analyzed in either of the two studies. we have analyzed the association between mthfr polymorphisms and chd by multiple methods in all genetic models and included more recent studies. to our knowledge, this is the first meta - analysis on association between mthfr polymorphisms and chd including both c677 t and a1298c. for the mthfr c677 t polymorphism, most studies have indicated that maternal c677 t was not a strong risk of chd, however, some reports have suggested its potential role in chd development. in our finding, no statistically significant difference was detected in genotype or allele frequencies of mthfr c677 t polymorphism in the mothers of chd patients compared with controls. only marginal association between maternal c677 t polymorphism and chd was found in recessive model. the finding was consistent with the previous studies involving both maternal and fetal c677 t polymorphism. particularly, we found a significant association between maternal c677 t and chd in recessive genetic models of asian subgroup, however, similar was not observed in caucasian. this discrepancy of association between asian and caucasian groups may be attributed to the different genetic and environmental factors. our have indicated that fetal mthfr c677 t polymorphism was significantly associated with chd in all genetic models. it was evident that fetal mthfr c677 t polymorphism was an important risk in the development of chd. to explain the , we speculated that decreased fetal mthfr enzyme activity may in a local hyperhomocystein environment, in which the heart could not develop normally (lu et al ., 2011). these evidences have supported the viewpoint that fetal mthfr c677 t polymorphism was more important than maternal mthfr c677 t polymorphism, and concentration of homocystein in fetus may influence heart development rather than maternal homocystein concentration. in addition, we found that fetal mthfr c677 t was significantly associated with chd in asian, while no statistically significant association was found in caucasian population. consistent with the from recessive model of maternal analysis, the fetal mthfr c677 t was more likely to be associated with chd in asian than caucasian. the have validated the notion that mthfr c667 t may be in combination with other genetic and environment factors to affect the fetal heart development. by considering the source of controls, the association between mthfr c667 t polymorphism and chd was significant in hospital based control group, though, not significant in population based control. the confounding from two subgroups categorized by source of control have indicated that hospital based and population based control was not homogenous in this study. we believe that the comparability between cases and controls contributes to the disagreement of these two subgroups. for the mthfr a1298c polymorphism, we found no statistically significant association between this polymorphism and chd either in maternal or fetal analysis. our finding has demonstrated that mthfr a1298c may not be a risk of congenital heart disease development. however, some studies indicated that the interaction between mthfr 1298 c allele and folic acid supplement increased the risk of having a child with chd (van driel et al . considering that minimal eligible studies included in our meta - analysis, this should be validated with more studied and large pooled samples in future . in subgroup analysis by ethnicity, only one study was performed in asian population to investigate fetal a1298c polymorphism and four studies were performed in caucasian population . because of the importance of mthfr polymorphisms in asian chd development, we suggest that more studies investigating association between a1298c polymorphism and chd be performed in asian population . although we made these findings in this meta - analysis, there were several limitations . first, our study was mainly based on unadjusted odd ratios, and the potential covariates including gender, age, vitamin supplement, smoking or other environmental factors, which might influence the final , were unable to control . we have investigated the study heterogeneity including geographic region, ethnicity, and source of control . however, none of them was identified as the potential source of heterogeneity between studies by meta - regression ( data not shown). we estimated that other unknown confounding factors may help explain the between - study heterogeneity. however, only a few studies included in our meta - analysis have classified their cases by types of chd. to analyze this issue despite the limitations mentioned above, the present study has demonstrated that the fetal mthfr c677 t polymorphism is an important risk of developing congenital heart diseases. our findings also suggest that mthfr a1298c polymorphism does not increase the susceptibility to chd. interestingly, we found that fetal mthfr c677 t polymorphism more likely affects asian fetus than caucasian fetus in the development of chd.

inconsistent were reported in recent literature regarding the association between methylenetetrahydrofolate reductase (mthfr) c677t / a1298c polymorphisms and the susceptibility of congenital heart disease (chd). in this study , we performed a meta - analysis to investigate the associations by employing multiple analytical methods.methodsliterature search was performed and published articles were obtained from pubmed, embase and cnki databases based on the exclusion and inclusion criteria. data were extracted from eligible studies and the crude odds ratios and their corresponding 95% confidence intervals (cis) were calculated using random or fix effects model to evaluate the associations between the mthfr c677t / a1298c polymorphisms and chd development. subgroup based analysis was performed by hardy weinberg equilibrium, ethnicity, types of chd, source of control and sample size.twenty-four eligible studies were included in this meta - analysis. significant association was found between fetal mthfr c677 t polymorphism and chd development in all genetic models. the pooled ors and 95% cis in all genetic models indicated that mthfr c677 t polymorphism was significantly associated with chd in asian, but not caucasian in subgroup analysis. the maternal mthfr c677 t polymorphism was not associated with chd except for recessive model. moreover, neither maternal nor fetal mthfr a1298c polymorphism was associated with chd.the fetal mthfr c677 t polymorphism may increase the susceptibility to chd. fetal mthfr c677 t polymorphism was more likely to affect asian fetus than caucasian. the mthfr a1298c polymorphism may not be a risk of congenital heart disease.

chest injuries constitute barely 34% of the consequences of blunt trauma, whereas nearly 25% of post - traumatic deaths are caused by injuries to the chest. such injuries are present in over half the patients after multifocal trauma. statistically, airway trauma is most commonly observed among men under 40 years of age, and the male - female ratio is 3: 1. 47%; nearly 80% of injuries are located less than 2 cm from the carina. left - sided bronchial injuries constitute 32% of cases, while only 2% involve trauma to both main bronchi. the rate of post - traumatic tracheal ruptures is around 19%; they are most often located in the cervical trachea (1015%). the nonspecificity of the clinical symptoms, which often overlap with symptoms arising from other injured organs, in diagnostic difficulties and delays the start of treatment. the symptom most often mentioned in clinical practice is subcutaneous emphysema; other frequently observed symptoms include dyspnea, changes of vocal timbre, hemoptysis, and bleeding (if the injury involves the bronchial arteries). imaging examinations reveal pneumothorax, sometimes bilateral. increased air leakage in the drainage system after the connection of a suction device to the pleural drain is an important symptom that suggests the possibility of airway disruption. in the case of injuries located in the thoracic part of the respiratory tract, mediastinal emphysema with the accumulation of air in the subcutaneous tissue may be the only symptom present. when airway injury is suspected, the primary diagnostic examination is bronchofiberoscopy, which also enables the assessment of the extent of the injury. in the case of intensified bleeding, classic rigid bronchoscopy is recommended. the patient was a 25-year - old male mine worker, who suffered a multiorgan injury when he was buried by falling rocks. on admission to the intensive care unit, mechanical ventilation was introduced along with pharmacological therapy with catecholamines and antibiotics. after a chest radiogram revealed right - sided pneumothorax and costal fractures (ribs ii v) the patient s crushed right foot was treated surgically (amputation at chopart s joint), while the fractured medial condyle of the femur was treated conservatively. due to the observed incidents of reduced respiratory fitness parameters, computed tomography of the chest the examination visualized irregularities in the outline of the left main bronchus and a consolidation of gas bubbles in this area, which prompted the suspicion of bronchial injury (figures 1 a, b, 2 a, b present a tomographic reconstruction of the respiratory tract). tomographic image reconstructions: deformation of the wall of the left main bronchus tomographic image reconstructions: vascular system and lung parenchyma diagnostic investigation was supplemented with bronchofiberoscopic examination, revealing hyperemia and massive edema of the mucosa in the proximal segment of the left main bronchus, immediately next to the tracheal bifurcation. consequently, the attempt to reach the left main bronchus with the endoscope was unsuccessful. the patient was referred for further treatment at the thoracic surgery center in wrocaw with a suspicion of airway disruption. 500 ml in volume ) and extensive hematomas in the tissues of the thoracic integuments. prepare of the mediastinal tissues revealed the completely amputated left main bronchus; the whole length of the membranous part of the bronchus was ruptured. subsequently the detached bronchus was anastomosed with the trachea, and the anastomosis was wrapped with an intercostal muscle flap. the patient s hospitalization continued at the intensive care unit of the lower silesian lung diseases center. , the drain from the left pleura was removed; 3 days later, the right pleural drain was removed as well. on the 6 day after the thoracotomy , the airway was reevaluated with an endoscope, revealing stenosis at the tracheobronchial anastomosis; the endoscope could not pass through this location. additionally, a large amount of mucous secretion, which filled the whole bronchial tree, was evacuated. consequently, the secretion accumulated again, ing in a repeated bronchoaspiration after less than 20 h. it was deemed necessary to perform tracheotomy. after the patient s cardiopulmonary parameters stabilized on the 14 postoperative day, the patient was referred for further treatment on the thoracic surgery department. the patient was discharged from the center on the 18 postoperative day. after less than 6 weeks , the patient was readmitted with symptoms of left main bronchus occlusion. for the past several days, the patient had manifested symptoms of airway infection: he was fever and he had dyspnea. an out - patient computed tomography (ct) scan demonstrated anastomotic stenosis (fig . obturation of the left main bronchus at the anastomosis site obstruction of the left bronchial tree with preserved lung aeration a chest radiogram ( fig . 5) taken on the day of readmission (7 days after the ct exam) revealed complete atelectasis of the left lung. radiological image of left lung atelectasis bronchofiberoscopic examination showed that the lumen of the left main bronchus was very narrow: its diameter did not exceed 3 mm. an attempt was made to perform mechanical dilatation, but without success. the diagnostic investigation was supplemented with chest ct, revealing obturation of the left bronchial tree (fig . computed tomography image : obstruction of the left bronchial tree computed tomography image : left lung atelectasis chest computed tomography : bronchographic reconstruction scans first postoperative day a decision was made to perform surgical intervention . thoracotomy was repeated with sleeve resection of the stenosed left main bronchus ( length : 11.5 cm), and the bronchus was anastomosed with the trachea (the posterior wall with single sutures, and the anterior wall with a continuous suture). figure 9 shows the chest radiogram obtained on the 1 postoperative day, while figure 10 presents a radiogram from the 6 postoperative day. chest x - ray from the day preceding the patient s discharge from the day of readmission, the patient was administered broad - spectrum antibiotic treatment. on the third postoperative day, the drain placed in the left pleura was removed. he continued to be monitored as an outpatient he attended follow - up visits, during which the site of anastomosis was evaluated with an endoscope. the first such examination took place 2 weeks after discharge, demonstrating permeability of the anastomotic site and no signs of inflammation. subsequent follow - up examinations took place every three months until the end of february, 2015. the final examination showed a stable endoscopic image with satisfactory width of the left main bronchial lumen. post - traumatic airway injuries are very rare in clinical practice. due to the character and multifocality of the traumatizing force, symptoms of other organs are often much more pronounced and mask the symptoms related to the respiratory system. the mortality rate is 60% for bilateral bronchial damage, 26% for tracheal injuries, and, respectively, 16% and 8% for isolated injuries of the right and the left bronchial tree. statistically, the median time until diagnosis is estimated at approximately 9 days, and until the start of surgical treatment at 25 days. the median time until diagnosis and treatment is, respectively, 30 and 42 days for left - sided bronchial injuries, 1 and 3 days for right - sided injuries, and 3 and 23 days for tracheal injuries. the type, extent, and possible consequences of a blunt trauma are determined by 3 factors: compression, truncation, and shock wave. , the energy is dispersed as the tissues buckle under compression; if the deformation is too quick, the tissues are ruptured. the first one assumes that the action of a violent compression force in the anteroposterior dimension in its transverse dispersion. consequently, the pulmonary parenchyma is pressed against the chest wall, and the lateral forces cause the lung to press on the tracheal bifurcation. this is presumably the dominant mechanism for crushing injuries. according to the second theory, confirmed in experimental canine studies, the rising compression of the chest and trachea with a closed glottis leads to a rapid increase of airway pressure (especially in the trachea and large bronchi). when the respiratory tract s elasticity is overcome, a rupture occurs (usually of the membranous part). the third theory assumes that during quick deceleration the force disperses, ing in traumatization of the trachea and the bronchial tree. this mechanism best explains the injuries occurring in traffic accident victims. injuries of the left bronchial tree are observed less frequently; this is due to the close proximity of the aortic arch, which stabilizes the bronchus. the force injuring the bronchus simultaneously causes a rupture of the aortic wall, which most often death on the scene. in the present case, the traumatic mechanism was probably somewhat different, as evidenced by the ct image and the intraoperative situation. the dynamism of the traumatic process was surprising in the context of the relatively inconspicuous clinical manifestation. the lack of sleeve resection of the injured bronchus during the first procedure is worth discussing. the decision was made in view of the patient s severe condition including cardiopulmonary failure, the impossibility of making technical provide for the anesthesiological and surgical team, and the intraoperative surprise factor. the literature features recommendations stating that reconstruction should be attempted in the case of bronchial injuries located proximally to the subsegmental branches, while the distal segments should be resected. the survival rates of patients treated with surgery are better than those of patients treated conservatively. the mortality rate is lower in patients in whom bronchial reconstruction was performed than in patients in whom the injured bronchial segment was resected with the peripheral fragment of pulmonary parenchyma (3% vs. 13%). in the case of tracheal injuries, the mortality rate was estimated at 6% in patients treated with surgery and 66% in patients treated conservatively. the present case raises the question whether covering the anastomotic site with a pedicled intercostal muscle contributed to the occurrence of stenosis. recommendations that can be found in the medical literature include the use of reversed parietal pleural flaps, pericardial fat, or fragments of the serratus anterior, pectoralis major, or latissimus dorsi muscle. in the long term, intercostal muscles show a tendency to calcify , as was the case in the present patient 9 weeks passed between the first and the second procedure. another question open for discussion is the possibility of dilatation the lumen of the stenosed bronchus with a laser. however, in view of complete lung atelectasis, the symptoms of infection, and the young age of the patient, a decision was made to perform surgical treatment. this case study was presented at the extended conference of the polish thoracic surgeon club (march 2021, 2015 in karpacz).

tracheobronchial damage is very rare in clinical practice and represents no more than 1% of all injuries caused by blunt trauma. nearly 80% of patients die before reaching the hospital. most ruptures are observed in the right main bronchus and are located within 2 cm of the carina trachea. the highest mortality rate applies to patients with bilateral bronchial injuries. nonspecific symptoms, additionally masked by complaints regarding other damaged organs, delay the diagnosis and surgical treatment. the aim of this article is to present one particular clinical case and to discuss it in conjunction with a literature review.

toll - like receptors (tlrs) are transmembrane receptors initiating a range of host defense mechanisms in response to microbial products. activation of the tlrs leads to activation of intracellular signaling pathways which in the production of inflammatory cytokines, or chemokines, inducing the development of antigen - specific adaptive immunity. these receptors recognize pathogen - associated molecular patterns as well as host - derived ligands released by various cell types during immune responses. this leads to signaling events ing in acute host responses necessary to kill the pathogens. this activation is beneficial for the host but can become deleterious if ing in chronic inflammation. beside their role in innate and adaptive immune responses, tlrs have been recently described to regulate energy metabolism, mostly through acting on adipose tissue. in particular, it was demonstrated that tlr4, the receptor for recognition of gram - negative bacterial cell wall components, was able to sense free fatty acids and to induce insulin resistance in adipose tissue. tlr2, as tlr4, has been shown to be activated by saturated free fatty acids and is implied in bacterial lipoprotein recognition. unlike other tlrs, which are functionally active as homodimers, tlr2 can form heterodimers with tlr1 or tlr6 that will recognize distinct molecular patterns of lipopeptides and can discriminate between tri- and diacylated lipopeptides. our purpose is to demonstrate that beside tlr4, tlr2 and its partners tlr1 and tlr6 are potentially susceptible to play also a role in adipose tissue inflammation. epidemiological studies have clearly demonstrated a relationship between intraabdominal fat depots and metabolic abnormalities related to obesity. in this regard, subcutaneous and omental adipose tissues display different metabolic features such as differences in lipolysis or adipokine secretion. since omental tissue is related to a higher degree of inflammation, a high expression of tlrs in this tissue may correspond to an implication of these receptors in obesity - related inflammation. we analyzed, tlr1, tlr2, tlr6, and tlr4 expression in paired human adipose omental and subcutaneous samples from subjects with different glycaemic status. we previously demonstrated that inflammation correlates with a decrease of lipogenesis and that tlr4 stimulation interferes with adipocyte differentiation. in order to determine whether tlr2 activation can be also implied in proinflammatory stimulation or interact with adipogenesis, we studied the impact of pam3csk4 (a tlr1/tlr2 agonist) exposure on 3t3-l1 preadipocyte cell line. insulin, dexamethasone and, isobutyl-1-methylxanthine were purchased from sigma chemical co. (st louis, mo, usa). the synthetic bacterial lipoprotein n - palmitoyl - s---cysteinyl--seryl--lysyl--lysyl--lysyl--lysine (pam3csk4) was from invivogen (san diego, ca, usa). tetramethylbenzidine (tmb) was from bd biosciences (franklin lakes, nj, usa). forty - one caucasian female subjects (seven lean volunteers and thirty - four obese patients) attending either the dpartement de chirurgie gnrale et endocrinienne, chru de lille or the department of endocrinology of the clnica universitaria de navarra were enrolled in the study. subjects were further classified into 3 groups according to the recently established diagnostic thresholds (based on an oral glucose tolerance test, ogtt) for diabetes and lesser degrees of impaired glucose regulation (normoglycaemia : fasting plasma glucose concentration ( fpg) < 100 mg / dl and 2-h pg < 140 mg / dl after ogtt; glucose intolerant: fpg > 100 mg / dl and < 125 mg / dl or 2-h pg between 140 and 199 mg / dl after ogtt; type 2 diabetes mellitus: fpg 126 mg / dl or 2-h pg 200 mg / dl after ogtt; table 1 ). the lean group included patients undergoing surgery due to benign diseases, such as cholecystectomy, while the 34 obese patients strictly met the criteria for bariatric surgery. in both groups of patients surgery informed consent was obtained from all subjects and the experimental design was approved by the hospitals' ethical committees responsible for research. biopsies were obtained from both subcutaneous and omental adipose tissues of lean and obese volunteers. 3t3-l1 preadipocytes were maintained and cultured in dmem (gibco, paisley, scotland, uk) containing 10% (vol / vol) fetal calf serum (gibco). 3t3-l1 cells were differentiated into adipocytes as previously described. briefly, 2-day postconfluent 3t3-l1 preadipocytes (designated day 0) were fed dmem containing 10% fcs, 10 g / ml insulin, 1 m dexamethasone, and 0.5 mm 3-isobutyl-1-methylxanthine for 2 days. cells were then fed dmem supplemented with 10% fcs and 5 g / ml insulin until day 10. human samples were homogenized using an ultra - turrax t25 basic equipment (ika werke gmbh, staufen, germany). total rna was extracted from 3t3-l1 cells or from human adipose samples using rneasy lipid kit (qiagen, courtaboeuf, france). one microgram of total rna was transcribed into cdna using cdna archive kit (applied biosystems, foster city, ca, usa). each cdna sample was analyzed for gene expression by quantitative real - time pcr (qpcr) using the fluorescent taqman 5-nuclease assay on an applied biosystems 7900ht sequence detection system. the taqman real - time pcr was performed using 2 taqman master mix and 20 premade taqman gene expression assays (applied biosystems). the mrnas levels were normalized to that of acidic ribosomal phosphoprotein (36b4), a gene whose expression is unaffected by adipogenesis. for human samples, glyceraldehyde-3-phosphate dehydrogenase (gapdh) was used as reference gene, since it was previously described to exhibit a low coefficient of variation and no significant differences in mrna levels between samples of the different phenotypical groups. the data are given as the ratio of the target gene mrna to that of gapdh or 36b4 mrna level. concentrations of cytokines and chemokines were measured by duoset elisa development system according to manufacturer instructions (r&d systems, abingdon, uk). to further confirm the specific tlr2 activation and to exclude potential endotoxin contamination of pam3csk4 agonist leading to tlr4 activation , we used a monoclonal anti - tlr2 antibody to inhibit its biological activity (clone t2.5 from hycult biotechnologies, uden, the netherlands, igg1 isotype). the negative control was performed using another igg1 monoclonal antibody (15h6, interchim, montluon, france). statistical analysis was performed with the spss software package (14.0.2, chicago, il). according to sample size , the test on ranks was performed and two - tailed exact p - values are given. the mrna levels between lean and obese patients or in cell culture studies were analyzed by u mann - whitney's test. comparisons of mrna levels between subcutaneous and omental adipose tissues were performed using the paired wilcoxon test. correlations between continuous variables were determined using the nonparametric spearman's rank correlation. the threshold of significance was set at p <.05. we first globally analyzed tlr1 and tlr2 mrna expression in omental and subcutaneous adipose tissue of 34 morbidly obese (bmi = 46.4 5.6 kg / m) and 7 lean subjects (bmi = 21.4 2.4 kg / m) by qpcr. we observed an increased expression of tlrs in omental fat compared to subcutaneous adipose tissue (1.42-, 1.35-, 1.35-, and 1.40-fold increase for tlr1, tlr2, tlr4, and tlr6, resp . ; the nonparametric paired wilcoxon test was performed between subcutaneous and omental values for each gene and demonstrated a significant difference . p - values are, respectively, tlr1 : 1,03 10 ; tlr2 : 3.84 10 ; tlr4 : 1,29 10 ; tlr6 : 2.30 10 . this difference was significant in obese normoglycemic and obese glucose intolerant groups, never in lean group and significant for tlr4 and tlr6 in diabetic subjects . we then performed spearman 's correlation analysis ( nonparametric analysis based on rank) between each tlr expression and each depot (data not shown). for a given tlr , expressions was strongly correlated in omental and subcutaneous adipose tissues (e.g., individuals with high tlr1 expression in the omental depot also showed a high subcutaneous tlr1 level). in each given depot, a correlation between the four tlrs expression was also observed (e.g., individuals with a high tlr1 expression in subcutaneous depot also display a high expression of all other tlrs in this depot). mann - whitney analysis was performed to detect an association between tlrs expression and glycaemic status. no correlation between expression levels of tlrs and glycaemic status was observed among obese subjects. the only significant value obtained was between lean and obese normoglycemic subjects in omental tissue for tlr1 and tlr2 (p = 3.73 10 and 3.58 10, resp .) to determine whether toll - like receptors are expressed and functional in an adipocyte cell line model, total mrna was isolated from 3t3-l1 preadipocytes as well as fully differentiated adipocytes, and qpcr was performed. considering differentiated adipocytes, the expression of tlr4 mrna was 76 times higher than that of tlr1, 4.6 times higher than that of tlr2 and 30 times higher than that of tlr6. this relative expression difference was also observed in preadipocyte but with a lower range (figure 3). differentiation into adipocyte has no significant impact on tlr2 and tlr6 expression levels while tlr1 expression is lowered (5-fold) and tlr4 is enhanced (4-fold). we wanted to demonstrate that, besides tlr4 as already published, other tlrs can be functional in 3t3-l1 cell line. we therefore stimulated 3t3-l1 preadipocytes and fully differentiated adipocytes with pam3csk4 (a tlr1/tlr2 agonist) and measured the expression level of inflammation markers by qpcr after 4 hours of induction. mrna coding for il6, ccl2, ccl11, nos2, ccl5, and ptgs2 was highly induced by pam3csk4 stimulation in preadipocytes as well as in fully differentiated adipocytes (figure 4). to further demonstrate the specificity of this stimulation and exclude the eventuality of tlr4 stimulation via endotoxin contamination a monoclonal antibody of the same isotype was used as a control and demonstrated no effect on the pam3csk4 induction. therefore, the stimulation of proinflammatory markers by pam3csk4 can specifically be attributed to tlr2 (figure 5). cell supernatants were collected and cytokine and chemokine secretions were measured by elisa at different time points after pam3csk4 stimulation. preadipocytes were mainly responsible for release of il6, whereas the ccl2, ccl5, and ccl11 concentrations observed were in the same range for both preadipocytes and adipocytes (figure 5), demonstrating that adipocytes are able to secrete proinflammatory products via tlr2/tlr1 activation in both states of differentiation. we previously demonstrated that tlr4 stimulation by lps was able to impair adipocyte differentiation of 3t3-l1 cells. to demonstrate that tlr2/tlr1 is also able to interfere with this process, we added 1 or 10 ng / ml pam3csk4 to the differentiating medium throughout the differentiation process. microscopic observation of lipid - laden cells stained with oil - red - o showed a slight reduction of lipid droplets in 3t3-l1 cells cultured with pam3csk4 at day four. since ppar is a key regulator of adipocyte differentiation, we monitored its expression level during the differentiation process. ppar mrna was detected 2 days after onset of differentiation and was further elevated at days 6 and 10. treatment with pam3csk4 led to a 50% reduction of ppar expression (figure 6), demonstrating that tlr2/tlr1 activation impairs adipogenesis. obesity is defined as a low - grade chronic inflammatory disease associated with a moderate increase of circulating inflammatory factors. this inflammation causes or worsens insulin resistance in insulin - responsive tissues such as adipose tissue, muscle, or liver. one of the causative factors of this inflammation process is the adipose tissue itself via its early infiltration with immune cells (mainly macrophages) and via its autocrine and paracrine secretion of pro - and anti - inflammatory cytokines. importantly, it was shown that macrophage infiltration is more prominent in visceral fat than in subcutaneous fat , thereby reinforcing the notion that intra - abdominal fat amount and metabolic abnormalities are correlated, as clearly shown by several epidemiological studies. one member of the tlr family, namely tlr4, was reported to participate to the development of inflammation and insulinresistance at the adipose level. we thus hypothesized that other functional members of the tlr family of innate immune receptors might also participate in these processes. since, like tlr4, tlr2 was demonstrated to sense fatty acids when dimerized either with tlr1 or tlr6 we first focused our search on this receptor and on its dimerization partners tlr1 and tlr6. we found that tlr4, tlr1, tlr2, and tlr6 are significantly overexpressed in omental adipose tissue. since a preferential macrophage infiltration into obese omental versus subcutaneous fat was demonstrated , we can not exclude that toll - like receptors expression in adipose tissue is mainly due to macrophages as suggested by others, but has to be demonstrated since many other adipose tissue cell types can express toll - like receptors. we observed a strong correlation between each tlr expression within a given adipose depot and that at the individual level, each tlr expression is correlated positively in subcutaneous and omental depots. , a cross - regulation between tlr4 and tlr2 expression was demonstrated after activation of 3t3-l1 cells with lps, a tlr4 agonist. whether lps is the effector signal triggering overexpression of tlrs in human omental tissue could be hypothesized but other tlr ligands could also be involved. endotoxemia was shown to participate in the initiation of obesity and insulin resistance, therefore serum lps concentration as well as free fatty acid concentration would be interesting to be evaluated in relation to tlrs expression. recently, resistin was demonstrated to be able to bind to tlr4, and other endogenous ligands such as hmgb1 or hyaluronan fragments were also reported as tlr4 or tlr2 activators. whether different tlr activators in obesity are of endogenous or exogenous origins or even of both, remained to be investigated and will be crucial for a better comprehension of this chronic inflammation. we previously demonstrated that the inflammatory state associated with a decreased expression of lipogenic markers was more pronounced in diabetic subjects. we could not reveal any differential expression according the prediabetic or diabetic status of the subjects. this might suggest that omental overexpression of toll - like receptors could play a role in early prediabetic phases of metabolic syndrome acquisition but not in further complications. activation of tlr2 by saturated fatty acids leads to the activation of myd88-dependent signaling pathways whatever heterodimer is implied. we here demonstrate that proinflammatory products can be synthesized by 3t3-l1 cells following tlr2/tlr1 activation. we have used the well - known 3t3t - l1 murine preadipocyte cell line as an in vitro system for adipocyte generation to study the inflammatory response of both preadipocytes and adipocytes upon tlrs stimulation. the expression level of tlrs in 3t3-l1 cell line has not to be compared to what was obtained from human samples since adipose tissue is constituted of numerous different cell types (e.g., adipocytes, macrophages, and endothelial cells). our purpose was to stress out the potential responsiveness of the adipocyte or of its precursors toward stimulatory compounds of the tlr2 pathway. we had already shown that il-6, certain chemokines (ccl2, ccl2, and ccl11), inducible nitric oxide synthase (nos2), and cyclooxygenase-2 (ptgs2), all important mediators of inflammation, can be induced via tlr4 activation in the 3t3-l1 cell line. tlr4 activation induced a lower secretion of il6, ccl5, and ccl11 in adipocytes when compared to preadipocytes, while ccl2 secretion was similar in both differentiation states. following tlr2 activation, only the secretion of il-6 was lower in differentiated adipocytes than in preadipocytes. similarly, we observed different induction factors for mrna levels of the proinflammatory enzymes nos2 and ptgs2 in this study of activation via tlr2 and in the previous study of activation via tlr4. recently, an exhaustive study of tlr1 to tlr9 activation of adipocyte with corresponding ligands was reported demonstrating a distinct response for each receptor. as stated before , the pertinence of these observations will be strengthened when the in vivo genuine ligands will be described. we have observed that tlr2 and tlr6 expression is constant throughout differentiation of 3t3-l1 cells, whereas tlr1 and tlr4 expression is modulated in fully differentiated adipocytes. whether this could explain the weaker secretion of il-6 observed in fully differentiated adipocyte can be hypothesized. we suggest that a fine regulation of the synthesis of proinflammatory mediators could be achieved in adipose tissue via differential expression and activation of tlr family members. as for tlr4 stimulation this is in agreement with previous observation that showing tnf release in human adipose tissue is mainly due to nonfat cells. to conclude, we demonstrate here that tlr2/tlr1 activation is able to interfere with adipocyte differentiation in the 3t3-l1 cell line, as previously described for tlr4. this could occur either directly or via secretion of adipoctye - derived proinflammatory and antiadipogenic products. this observation is in agreement with our previous study demonstrating that the expression of lipogenic factors is reduced in omental adipose tissue in correlation with inflammation increase. beside their defense function of alerting the immune system of the presence of pathogenic microorganisms, tlrs can also sense dietary lipids therefore, it is tempting to speculate that detection of abnormal level or composition of these lipids will induce a physiological response. our observations suggest that it might be the case and that, in addition to tlr4, other tlr family members, that are functional and present in adipocytes, could play this role. unfortunately, the physiological response in elicitation of a chronic omental adipose inflammation which contributes to metabolic syndrome.

it was recently demonstrated that tlr4 activation via dietary lipids triggers inflammatory pathway and alters insulin responsiveness in the fat tissue during obesity. here , we question whether other tlr family members could participate in the tlr - mediated inflammatory processes occurring in the obese adipose tissue. we thus studied the expression of tlr1, tlr2, tlr4, and tlr6 in adipose tissue. these receptors are expressed in omental and subcutaneous human fat tissue, the expression being higher in the omental tissue, independently of the metabolic status of the subject. we demonstrated a correlation of tlrs expression within and between each depot suggesting a coregulation. murine 3t3-l1 preadipocyte cells stimulated with pam3csk4 induced the expression of some proinflammatory markers. therefore, beside tlr4, other toll - like receptors are differentially expressed in human fat tissue, and functional in an adipocyte cell line, suggesting that they might participate omental adipose tissue - related inflammation that occurs in obesity.

magnetic resonance imaging (mri) gives superior imaging resolution and is increasingly the modality of choice for functional and anatomical imaging in cardiovascular disease. in an aging population the prevalence of patients with implanted trans - venous pacing systems means that, increasingly, such patients will require a cardiac mri study; however, the presence of a permanent pacemaker (ppm) has traditionally excluded mri as a viable imaging modality. the use of mri as a viable and safe imaging modality for individuals with an implanted ppm has been an issue of debate for some time. however, there are reports of hazards both to the device and the patient, including fatalities. historically, the main concerns centered around the potential for the strong magnetic fields to move the device, cause inappropriate pacemaker stimulation, potential alterations to the device programming, and to create cardiac tissue damage through local heating with consequent alteration in lead thresholds. confidence has improved following the recent advent of mri conditional systems, approved and ce marked for conditional use with mri scanning, including cardiac mri. whilst mri scanning of anatomy remote to the heart has been increasingly reported , there remains a reluctance to use mri fields to directly image the heart and thorax. indeed, initial recommendations involved keeping the pacing device away from the isocenter of the magnet, hence precluding cardiac imaging. the concern was that the risk of damage to the device or the patient would be too high if the magnetic field was concentrated directly over the heart, as well as the increased potential for artefact attenuation of image quality. however, despite growing evidence to support the safety of these devices in mri scanning of the heart under experimental conditions, to our knowledge there are no published reports regarding their use with cardiac mri in a clinical setting. a 64-year old man presented with a history of increasing exertional breathlessness, chest tightness and intermittent fatigue shortly after undergoing left atrial ablation for paroxysmal atrial fibrillation (af). following an initial diagnosis of af in 2005, medical management with anti - arrhythmic drugs (including amiodarone) had been effective, but worsening symptoms necessitated left atrial ablation with pulmonary vein isolation in 2009. subsequent to this, he reported increasing breathlessness and chest discomfort. due to persistent symptomatic sinus bradycardia given his ongoing symptoms and the uncertainty of the underlying diagnosis, the pacing system selected for implant was a medtronic enrhythm mri surescan dual chamber device and capsurefix mri leads (figure 1). this pacing system (generator box and leads) is the first to be designed to be compatible with mri scanning under pre - defined conditions; in general terms, these conditions include marginal limitation of the magnetic field and ensuring that the pacemaker is well established and functioning reliably. radio - opaque labels are present on the lead (large circle) and device (smaller circle) to indicate that the device can be used with magnetic resonance imaging. chest x - ray, enlarged image of generator box. radio - opaque labels are present on the lead (large circle) and device (smaller circle) to indicate that the device can be used with magnetic resonance imaging. the patient's past medical history included wolf - parkinson - white syndrome, with successful ablation of a right free wall accessory pathway in 1995, moderate aortic valve regurgitation, essential hypertension, benign prostatic hyperplasia, obesity (bmi=37) and mild psoriasis. physical examination revealed a regular paced rhythm, quiet aortic stenotic and regurgitant murmurs, and clear lung fields. trans - thoracic echocardiography provided non - diagnostic images as a consequence of body habitus. coronary angiography four years previously had demonstrated no significant coronary disease. in view of the wide differential diagnosis and the patient's previous exposure to high - dose ionising radiation, mri scanning was considered the most appropriate imaging modality to gather information on coronary perfusion, left ventricular function, valvular status, pericardial constraint and pulmonary venous anatomy in a single imaging procedure, free of further ionising radiation. the scan was performed according to a local protocol designed to closely follow the conditions of use published by the manufacturers of the device. close liaison with the manufacturers' technical representatives was maintained throughout. a 1.5 t magnetic field (philips intera, philips healthcare, the netherlands) was employed and specific absorption rate (sar) was kept to 1 watts / kg or less; well beneath below the 2 watts / kg advised. the device pocket had healed well over the previous five months and lead thresholds were stable, comfortably below the stipulated capture thresholds of 2.0 volts at 0.4 millisecond pulse width (table 1). as the patient was not dependent on pacing , the device was programmed to the manufacturers' advised setting of odo (i.e. sensing only, not pacing) during the scan, with continuous non - invasive hemodynamic monitoring. the device sensing and pacing parameters pre- and immediately post - scan did not change to any significant degree (table 1). subsequent pacing checks also proved unremarkable, with no undue changes to battery longevity. table 1characteristics of the pacing leads before and immediately after the scan.pre-scanpost-scanatriumventricleatriumventriclethreshold @ 0.4 ms (v)0.5111sensing (mv)3.37.83.97.6impedance (nms)416480440528 diagnostic quality images were acquired, including first pass adenosine stress perfusion imaging, and early and late gadolinium enhanced imaging. a minor degree of artefact was reported due to dephasing (figures 2 and 3) which did not compromise the quality of data interpretation. figure 2still images taken from balanced steady state free precession (bssfp) cine image acquisition. trans - axial view of the heart (left hand panel) showing lead artefact (arrow) in the right atrium (ra) and right ventricle (rv). trans - axial view of the pre - pectoral pocket (right hand panel) showing artefact from the pacemaker generator box (arrow): artefact did not compromise the quality of data interpretation. still images taken from balanced steady state free precession (bssfp) cine image acquisition. trans - axial view of the heart (left hand panel) showing lead artefact (arrow) in the right atrium (ra) and right ventricle (rv). trans - axial view of the pre - pectoral pocket (right hand panel) showing artefact from the pacemaker generator box (arrow): artefact did not compromise the quality of data interpretation. figure 3(a) four chamber orientation of the heart using black blood imaging showing a small amount of artefact in the right atrium. (b) still image from a bssfp cine clip showing excellent resolution with only minimal artefact. (c) late gadolinium enhanced imaging showing absence of left ventricular scarring, again with minimal interference from the pacing leads. (a) four chamber orientation of the heart using black blood imaging showing a small amount of artefact in the right atrium. (b) still image from a bssfp cine clip showing excellent resolution with only minimal artefact. (c) late gadolinium enhanced imaging showing absence of left ventricular scarring, again with minimal interference from the pacing leads. the scan comprehensively evaluated both anatomy and function without the need for ionising radiation and without compromising the image quality. the scan was performed according to a local protocol designed to closely follow the conditions of use published by the manufacturers of the device. close liaison with the manufacturers' technical representatives was maintained throughout. a 1.5 t magnetic field (philips intera, philips healthcare, the netherlands) was employed and specific absorption rate (sar) was kept to 1 watts / kg or less; well beneath below the 2 watts / kg advised. the device pocket had healed well over the previous five months and lead thresholds were stable, comfortably below the stipulated capture thresholds of 2.0 volts at 0.4 millisecond pulse width (table 1). as the patient was not dependent on pacing, the device was programmed to the manufacturers' advised setting of odo (i.e. sensing only, not pacing) during the scan, with continuous non - invasive hemodynamic monitoring. the device sensing and pacing parameters pre- and immediately post - scan did not change to any significant degree (table 1). subsequent pacing checks also proved unremarkable, with no undue changes to battery longevity. table 1characteristics of the pacing leads before and immediately after the scan.pre-scanpost-scanatriumventricleatriumventriclethreshold @ 0.4 ms (v)0.5111sensing (mv)3.37.83.97.6impedance (nms)416480440528 diagnostic quality images were acquired, including first pass adenosine stress perfusion imaging, and early and late gadolinium enhanced imaging. a minor degree of artefact was reported due to dephasing (figures 2 and 3) which did not compromise the quality of data interpretation. figure 2still images taken from balanced steady state free precession (bssfp) cine image acquisition. trans - axial view of the heart (left hand panel) showing lead artefact (arrow) in the right atrium (ra) and right ventricle (rv). trans - axial view of the pre - pectoral pocket (right hand panel) showing artefact from the pacemaker generator box (arrow): artefact did not compromise the quality of data interpretation. still images taken from balanced steady state free precession (bssfp) cine image acquisition. trans - axial view of the heart (left hand panel) showing lead artefact (arrow) in the right atrium (ra) and right ventricle (rv). trans - axial view of the pre - pectoral pocket (right hand panel) showing artefact from the pacemaker generator box (arrow): artefact did not compromise the quality of data interpretation. figure 3(a) four chamber orientation of the heart using black blood imaging showing a small amount of artefact in the right atrium. (b) still image from a bssfp cine clip showing excellent resolution with only minimal artefact. (c) late gadolinium enhanced imaging showing absence of left ventricular scarring, again with minimal interference from the pacing leads. (a) four chamber orientation of the heart using black blood imaging showing a small amount of artefact in the right atrium. (b) still image from a bssfp cine clip showing excellent resolution with only minimal artefact. (c) late gadolinium enhanced imaging showing absence of left ventricular scarring, again with minimal interference from the pacing leads. the scan comprehensively evaluated both anatomy and function without the need for ionising radiation and without compromising the image quality. to our knowledge, this is the first report in a clinical setting of the safe use of cardiac mri to investigate a patient previously implanted with an mri conditional pacemaker. only minor adjustments were required to the usual scanning protocol and high quality diagnostic images were readily obtained. importantly, the patient experienced no ill effects and there was no change in pacemaker function. whilst there have been reports of mri scans undertaken on various parts of the anatomy, including the heart, of individuals with pacing devices, we demonstrate that safe high quality cardiac mri scanning with a dedicated mri conditional pacemaker device can be performed safely and successfully in a clinical setting.

cardiac magnetic resonance imaging (mri) is increasingly used as the optimum modality for cardiac imaging. an aging population and rising numbers of patients with permanent pacemakers means many such individuals may require cardiac mri scanning in the future. whilst the presence of a permanent pacemaker is historically regarded as a contra - indication to mri scanning, pacemaker systems have been developed to limit any associated risks. no reports have been published regarding the use of such devices with cardiac mri in a clinical setting. we present the safe, successful cardiac mri scan of a patient with an mri - conditional permanent pacing system.

intra - articular knee injection is a common, relatively simple and safe procedure done in an outpatient setting for various knee conditions. injections of corticosteroids and hyaluronic acid (ha) are most common for the treatment of knee osteoarthritis (oa). although several portals are available for knee injection12 ), each has its own advantages and disadvantages. accuracy of intra - articular needle placement, portal site pain and experience of the clinician are important factors to be considered before selecting a portal for knee injection. the two routinely used approaches for intra - articular knee injections are superolateral and anterolateral. the superolateral approach with the leg in extension the anterolateral approach is familiar among knee surgeons due to its routine use in arthroscopic surgery. it allows the patient to remain in a sitting position with the knee bent, and bilateral injections can be performed with ease without changing the patient's position. it is, therefore, useful in patients whose knee can not be extended, and furthermore, it does not require manipulation of the patella4 ). accordingly, most of the previous studies stressed upon the accuracy of intra - articular needle placement. few studies have described procedural pain and the degree of pain relief following intra - articular knee injection through various portals. hence, we sought to determine whether injection through the anterolateral portal provokes less pain and provides better pain relief than the superolateral portal. this prospective randomized controlled study was undertaken in 60 patients from 30 june 2014 to 30 october 2014 at our tertiary care center. patients were adequately educated regarding the nature of the study before the procedures. written informed consent and clearance from the local ethical committee were obtained before the initiation of the study. inclusion criteria were patients with radiological kellgren - lawrence grade ii or iii oa knee and ability to give informed consent. exclusion criteria were as follows: conditions other than primary oa, systemic diseases that may affect the , ha and steroid injections within recent three months, allergy to ha injection, the use of warfarin or antiplatelet therapy, or the presence of any infection. patients were recruited in our outpatient department and randomly assigned to either the superolateral injection group or the anterolateral injection portal group using computer - generated permuted block randomization. there were 29 patients in the superolateral group and 31 patients in the anterolateral group. demographic features and preoperative status of the patients including the oa severity in the patellofemoral and tibiofemoral joints and mechanical tibiofemoral angle were compared between the two groups (table 1). underlying knee pain was recorded using visual analogue scale (vas), where 0 cm = no pain and 10 cm = unbearable pain67 ). all patients received weekly injections for three weeks in accordance with the assigned route on an outpatient basis under strict aseptic precautions. all procedures were carried out using 23 gauge needles blindly based on anatomic landmarks by an experienced surgeon. injection through the anterolateral portal was given with the patient in a sitting position with the knee flexed to 90 at 1 cm proximal to the joint line, lateral to the patellar tendon, the needle was directed towards the intercondylar notch (fig . injection through the superolateral portal was performed with the patient in supine position . with the knee extended, the needle was inserted 1 cm above and 1 cm lateral to the superolateral margin of patella at a 45 angle in the cephalolateral to caudomedial direction ( fig . the first injection included ha ( 20 mg) and triamcinolone (40 mg) and subsequently, the second and third injections included only ha (20 mg). the primary outcome variable was the degree of pain measured at the portal site during the first injection, and the secondary outcome variable was the degree of pain relief, which was evaluated at 4 weeks after the last injection. procedural pain and knee pain were evaluated using a 0 - 10 vas, where 0 indicates no pain and 10 indicates the most severe pain. in order to ensure the validity and reliability of pain evaluation, a single investigator (lee sy) assessed pain levels for all patients. 20.0 (ibm co., armonk, ny, usa) and p - values of < 0.05 were considered statistically significant. the chi - square test was used to compare categorical variables, and the student t - test or paired t - test was used to compare numerical variables. injection through the anterolateral portal provoked less pain than the superolateral portal, and no differences in the degree of pain relief at 4 weeks after last injection were found between the two groups. the mean procedural pain was lower in the anterolateral group than in the superolateral group (1.5 vs. 2.7, p=0.004) (table 2). no intergroup differences were found in pain level (2.9 vs. 3.1, p=0.517) or the degree of pain relief (2.3 vs. 2.2, p=0.883) at 4 weeks after last injection. there were no immediate complications noted following injection, such as transient flushing reaction or erythema at the injection site. intra - articular injection of the knee joint is commonly performed in clinical practice and is the most common invasive procedure in sports medicine4891011 ). although the accuracy of the intra - articular needle placement through various routes has been vastly studied, pain - related factors such as procedural pain were barely touched in the literature. the present study hypothesized that knee injection through the anterolateral portal would be less painful and provide better short - term pain relief than the superolateral portal. we found that injection through the anterolateral portal provoked less pain than the superolateral portal, and no differences in the degree of pain relief at 4 weeks after last injection were found between the two groups. findings in this study supported our primary hypothesis that injection through the anterolateral portal would provoke less pain as compared to the superolateral portal. our findings are in contrast with a previous study reporting no significant difference in procedural pain between the modified anterolateral and lateral mid parapatellar portals12 ). on the contrary, our findings are in line with another previous study on the anterior approach for knee arthrography, where significant reduction was observed in absolute and relative degree of pain for the anterolateral route compared with the anterior paramedian route5 ). we speculate that thinner soft tissue for the needle to transverse may be related to less pain in injection through the anterolateral portal, particularly when the knee is flexed to 90. in addition, pain detected during the superolateral approach can be explained by accidental needle collision with the bone, quadriceps tendon, and suprapatellar synovium13 ). nonetheless, our study does not contain any data explaining why and how injections through the anterolateral portal provoke less pain than through the superolateral portal. future studies are warranted to scrutinize this issue. in our study, the degree of pain relief at 4 weeks of follow - up was comparable between the two groups. this does not support our secondary hypothesis that injections through the anterolateral portal offer better pain relief than through the superolateral portal. there are only two studies that compared clinical outcomes between different knee injection sites, and both studies found no significant difference between each other: lateral mid patellar injection vs. anterolateral injection12 ) and infrapatellar injection vs. medial knee injection14 ). on the other hand, several studies compared clinical outcomes of ultrasound - guided versus blinded injections and reported that ultrasound - guided injections provided better short - term clinical outcomes than blinded intra - articular knee injections1516 ). however, as the ultrasound - guided injection technique requires expensive devices and trained skills, its clinical and practical values should be evaluated according to the situation of each physician. first, the accuracy of intra - articular needle placement was not confirmed. before the initiation of this study , we considered ultrasonographic or radiographic confirmation of the accuracy of intra - articular needle placement, but it deemed impractical or unethical to expose study participants to additional expense or radiation hazards. furthermore, we noted that even though the use of needle guidance might improve the accuracy of knee injections, insufficient evidence existed to prove that increased accuracy of knee injections would lead to improved therapeutic outcome. a previous study reported that blinded knee injections were reasonably accurate in the lateral injection sites5 ). a recent systematic review found that overall one in five blinded knee injections were inaccurate17 ). in the systematic review, pooling data across studies suggested blinded knee injection at the superolateral portal site was most accurate (87%) while injections through medial mid - patellar portal (64%) and anterolateral portal (70%) were less accurate. therefore, whether injection through the anterolateral portal is more accurate than injection through the superolateral portal should be elucidated in future studies. the of this study may have been affected by the experience of the surgeon, which may limit generalization of our findings. hence, studies evaluating the degree of pain through portals of different approaches are required. finally, this study should be regarded as a preliminary study using a small sample size, which prompts future studies with larger sample sizes and sophisticated evaluation tools regarding needle placement accuracy. because of the small sample size, we could not perform subgroup analyses according to various factors that could influence the technical difficulty during injection or the degree of pain relief such as body mass index, oa, severity, and the presence or severity of patellofemoral joint. therefore, we could neither mention the effects of the confounders nor recommend individualized portal selection. it provokes less pain and provides better short - term pain relief than the superolateral portal. randomized trials to evaluate pain upon multiple routes of injections as well as accuracy of needle placement are needed.

purposeintra - articular knee injections are commonly performed in clinical practice for treating various knee joint disorders such as osteoarthritis and rheumatoid arthritis. when selecting the portal for injection, not only intra - articular needle accuracy but also procedural pain should be taken into consideration. the purpose of this study was to determine whether injection through anterolateral portal provokes less pain and provides better pain relief compared to superolateral portal.materials and methodsa total of 60 patients with primary osteoarthritis of the knee receiving intra - articular injections were randomized into 2 groups according to the type of portal approach; anterolateral or superolateral. all patients received hyaluronic acid (20 mg) and triamcinolone (40 mg) as the first injection followed by second and third injections of hyaluronic acid on a weekly basis. underlying knee pain, procedural pain, and knee pain at 4 weeks were evaluated using visual analogue scale (vas).injection through anterolateral portal provoked less pain (vas, 1.51.3) than the superolateral portal (vas, 1.5 vs. 2.7 ; p=0.004). no differences were found in the degree of pain relief at weeks between the two groups (p=0.517).we recommend the use of anterolateral portal for intra - articular knee injection as it provokes less pain and comparably short - term pain relief than the superolateral portal.

written informed consent was obtained from the patient for publication of this case report and accompanying images. a copy of the written consent is available for review by the editor - in - chief of this journal on request. yes, the guarantor accepts full responsibility for the work and conduct of the study, had access to the data, and controlled the decision to publish.

highlightsgenitalia are a key element for male self - esteem, contributing towards mental and physical balance and influencing their social life.scrotal dartos - fascio - mio - cutaneous flap for shaft coverage offers a large amount of tissue with low donor site morbidity, avoiding unsightly scars in more visible parts of the body.

how should patient categories, or case - mix groups, be defined if they are to be used for prospective payment for inpatient care? two criteria are important for evaluating the effectiveness of a case - mix grouping system: medical meaningfulness of the grouping system and homogeneity of resource consumption within each of the case - mix groups. in addition, if a case - mix grouping system is to be used for prospective payment, two additional criteria must be considered: independence from the medical treatment process, and administrative feasibility. with these criteria in mind, a severity of illness index was developed by a team of researchers, physicians, and nurses at the johns hopkins university. it was designed to be a medically meaningful generic classification system that could differentiate the severity of illness of hospital inpatients. because discharge abstract data contain only labels of principal and secondary diagnoses, procedures performed, age, etc. , the conventional discharge abstract data base is not rich enough to describe patient severity of illness accurately. using the patient's total medical record, the severity of illness index assigns to each patient at discharge an overall severity score that is determined from the scores of each of seven medically meaningful dimensions chosen to reflect burden of illness. these seven dimensions, as shown in figure 1, are: stage of the principal diagnosis the severity of illness index is described in detail in various reports (horn, sharkey, and bertram, 1983 ; horn, 1983a ; horn and sharkey, 1983 ; horn, chachich, and clopton, 1983 ; horn, 1983b). to determine the severity of illness score for an individual case, a rater scores each of the seven dimensions into one of four levels of increasing severity by examining data in the patient's medical record following discharge. definitions of each of the four levels for each dimension are provided, and raters are intensively trained to distinguish the reasons for selecting the level of each dimension. the rater then assigns an overall severity score for the patient on a four - point scale by implicitly integrating the values of the seven dimensions. the reliability and validity of the severity of illness instrument has been studied in several ways. more than 95 percent of the individual raters achieve greater than 90 percent agreement on blind re - rating of a sample of their charts after using the severity of illness index for 2 months. all the hospital data reported here had greater than 90 percent agreement rates on blind re - rating of samples of charts. the actual agreement percent, rather than agreement corrected for chance agreement, has been reported here because of the very different distributions of overall severity of illness in the samples of charts selected for blind re - rating. we did not feel that each rater's reliability assessment should be affected because the random sample selected for that review happened to contain charts from mostly one of the severity of illness levels. however, over 90 percent of the time, the agreement percent and the agreement corrected for chance agreement statistic were within 2 percentage points of each other. thus, we found that severity of illness data can be collected in a reproducible manner. with respect to the validity of the severity of illness instrument, we used the following procedures to assure that the instrument would come as close as possible to accurately measuring patient severity of illness. content validity was assessed by presenting the criteria for classification (definitions of severity levels) to a panel of medical experts who systematically examined the definitions and came to a consensus. the clinical experts felt that the criteria were representative of patient severity of illness at each identified severity level. face validity was examined by asking the developers and users of the instrument to make subjective judgments as to whether or not the instrument seemed reasonable and had the ability to obtain reasonable data. empirical investigation suggests that the index is a good measure of severity, but the process of validation is constantly being examined. the severity of illness index can not be compared with another validated measure of severity because none is available at this time. the research and statistical analyses discussed below, however, present evidence that the instrument leads to an accurate measurement of illness severity. the four levels of severity are an ordinal scale from the least severe (level 1) to the most severe (level 4); the distances between successive levels have no significance and are not necessarily equal. the severity of illness index is a generic measure, referring to the patients themselves. thus, any grouping system for patient classification can be subdivided into severity of illness levels. in particular, the severity of illness index can be used within drg's or within any other case - mix system. another feature of the severity of illness index, which it shares with other case - mix grouping systems such as drg's and disease staging, is that it does not explicitly take into account the quality of care received by the patient. the severity of illness index asks how sick the patient is and what burden of illness the patient exhibits while in the hospital. it does not ask whether the patient's burden of illness was naturally caused or iatrogenically caused, (i.e., was exacerbated by accident or infection during hospitalization). thus, a patient may become sicker as a of poor quality of care. severity of illness data are being or have been collected in more than 30 hospitals in the united states and canada. these hospitals include university teaching hospitals, community teaching hospitals, and community nonteaching hospitals. the presented here are based on those hospitals for which we had at least six months of data. these are preliminary and will need to be studied more thoroughly when additional data become available. because of the controversies about what trim points should be used, the data have not been trimmed. this permits generalization to an entire patient population, and thus application of a prospective payment system to all patients. severity of illness data are usually collected at discharge by medical records personnel concurrent with the coding of discharge abstract data, or by utilization review personnel when a final utilization review on a patient is completed. for analysis, severity of illness data are merged with discharge abstract data and financial data on each patient. at this point, a computer algorithm takes the presence of an operating room procedure into account. the data are subdivided into three subgroups depending on whether the patient had no operating room procedure, a moderate operating room procedure, or a major operating room procedure. the designation of moderate versus major operating room procedure is based on a list developed by a surgeon panel. a major operating room procedure either requires very special skills and education or takes a long time from which to recover. the subdivision into procedure type subgroups is done for analytical purposes, but does not affect the patient's severity rating. within each severity of illness group, we examined resource consumption as expressed by total charges, length of stay, and ancillary service charges such as laboratory, radiology, pharmacy, and routine charges. we studied severity of illness within drg's and within major diagnostic categories (mdc 's), of which the drg's are a finer subdivision. the are similar for all types of resource use, so we shall demonstrate them using total charges. the necessary data are being collected to take charges back to costs, but the are not yet available. in figure 2, we show a sample of the reduction in variance (riv) obtained when patients are grouped within an mdc. the patients are placed either into drg's (from 5 to 48 subgroups depending on the mdc) or into the four severity levels divided into procedure type subgroups (up to 12 subgroups). is represented by the formula: the data come from a total sample of over 19,000 cases in one university teaching hospital. these are representative of the kinds of riv that have been seen for all the mdc's in the hospitals in the data set. the data show that even though the severity of illness index may place patients into fewer groups (at most 12) than the drg's, the variability in resource use that is explained by the severity of illness groups is greater than the variability that is explained by drg's. the weighted (by sample size) coefficient of variation statistics for the same mdc's are shown in figure 3. the coefficient of variation of a data set is the standard deviation divided by the mean. we see that the severity of illness groups have lower weighted coefficients of variation, and hence, by this measure, have greater homogeneity than the drg's. analysis of variance f test statistics for the severity of illness index and for drg's are shown in figure 4. the analysis of variance f test indicates how different the means are between the various groups, compared with the variability within the groups. a higher f value indicates a grouping system with greater differentiation among the groups as well as better homogeneity within the groups. we see that the severity of illness groups have much higher f values than the drg's. thus, by three common measures of homogeneity, the severity of illness groups, even though almost always fewer in number, are found to be more homogeneous with respect to resource use than drg's. these are representative and typical of those seen from all the hospitals collecting severity of illness data on an ongoing basis. homogeneity of resource use is a desirable feature of a case - mix grouping system for prospective payment. it implies that most cases in a group will actually have resource - use levels which are approximately that of the typical (average, median, or whatever) case in the group the norm used to determine the amount of prospective payment for all patients in the group. even if a particular hospital's patient distribution is atypical, it will be reimbursed appropriately at levels that fairly reflect the resource consumption of its patients. if groups are heterogeneous, the possibility exists that a hospital's patients in a group will cluster far from the typical a grouping system with more groups might be expected, in principle, to be able to produce groups that are more homogeneous than those produced by a system with fewer groups. for the drg's and severity of illness case - mix systems thus, it is not just the number of groups that has a decisive influence on homogeneity, but also the fundamental conception of what is being quantified by the system. the data indicate that just the labels of principal and secondary diagnoses, age, procedures, etc., on which drg's are based, do not describe sufficiently well how severely ill the patients are. it was found, however, that dividing patients into severity of illness groups yields much better explanatory power. an example of these for one hospital with more than 19,000 cases in 1 year is shown in figure 5. patients were placed into drg's and also classified by severity of illness level and procedure type alone, severity and procedure type within mdc's, and severity and procedure type within drg's. for each of these three ways to use severity of illness, the is that the severity of illness groups are more homogeneous than the drg's alone, as indicated by greater reductions in variance, larger f values, and lower coefficients of variation. the number of groups into which the patients were classified in this hospital for each of the case - mix grouping systems is given in parentheses in the legend. in tables 1 - 3 , examples are shown of three drg's, each illustrating a different aspect of variability. table 1 is for drg 75 (major chest procedures), which contained 47 patients whose total charges ranged from $ 1,117 to more than $ 205,000. a patient was placed in the major operating room procedure category if any of the patient's operations was on the major operating room procedures list. otherwise, the patient was placed into the moderate operating room procedures category. in particular, the patient with charges of $ 1,117 was classified by the drg grouper into drg 75 because of a procedure coded 33.27 (other lung biopsy). our refined definitions of procedure codes do not list this procedure in the major category, nor does this patient's resource use reflect such a designation. however, some differences are noted in resource use by severity of illness level even among those patients who have only a moderate operating room procedure. the same phenomenon is observed for patients with major operating room procedures. large differences can be found between the average resource use for patients in the same severity level who have a moderate operating room procedure versus a major operating room procedure. the coefficients of variation of each of the severity of illness groups are much smaller than the coefficient of variation for drg 75 overall. by dividing the patients into severity of illness and procedure type groups within this drg, table 2 is for drg 108 (cardiothoracic procedures, except valve and coronary bypass, with pump), which contained 60 patients with total charges ranging from a little more than $ 5,000 to more than $ 289,000. although all these patients had a major operating room procedure, some differences in resource use by severity of illness level were found. table 3 is for drg 296 (nutritional and miscellaneous metabolic disorders, age over 70 and/or with a complicating or comorbid secondary diagnosis), which contained 52 patients and had a smaller range of total charges, from $ 374 to more than $ 57,000. however, differences were still observed in resource use as severity of illness increased. in this case, however, the severity level 1 group had a rather large coefficient of variation. this is attributable to one patient who had difficulty in placement, and whose resource use was therefore much higher than would be expected for a patient in this level. one of the main reasons for adopting a prospective payment system is to encourage an efficient level of operation within the health care delivery system. within a hospital , a prospective payment system should provide specific incentives for reducing inefficiencies of hospital services and should have groups that are meaningful to the medical community. if it does, then physicians may be led to modify their behavior, if appropriate. it is also important that a case - mix system for prospective payment produce groups that are homogeneous (by various measures) with respect to patient resource use. because of the often great variability in resource use found within drg's , a question arises about their effectiveness in inducing desirable modifications in physician practice patterns. severity of illness within drg's produced the most homogeneous groups (figure 5). how much of this variability might be explained by differences in physician practice patterns, the drg and severity of illness groups were subdivided further into groups of patients treated by each individual physician. we could, by this means, compare how much of the variability of charges within drg's could be explained by physician practice patterns, compared with how much of the variability of charges within severity - of - illness - adjusted drg's was explained by physician practice patterns. the of these comparisons are presented in figure 6 for several hospitals. in general, the show that drg's explain 30 - 40 percent of the variability in resource use. physician subgrouping within drg's explains another 20 - 40 percent of the variability of resource use. taking severity of illness into account as well, raises the explanatory power to between 90 percent and 96 percent of the variability in charges for each of the hospitals. these suggest that most of the variability in resource use within a hospital can be explained by: the case mix that is going to the hospital (drg). the coefficients of variation for these same hospitals grouped in the same ways are shown in figure 7. these show that physicians do explain some of the variability in resource use. however, it is not clear if the variability from different physicians treating the same type of patient differently (efficiency) or differences in patients within a drg, severity, procedure type group treated by the same physician (classification). these , as well as the contribution of poor quality of care and its effect on the definitions of severity of illness and drg's, need further study. if a hospital's (or a physician 's) resource use within a drg is higher than a typical level, it could be that the institution (or physician) is inefficient. it could also mean that the institution or physician is treating patients who are more severely ill. because severity - adjusted drg's produced the most homogeneous groups in this study, patients from the whole institution were placed into the appropriate drg group and further subclassified by severity of illness level and procedure type (drg, severity, procedure). as the norms of practice in each institution, we used the average resource use expressed in terms of total charges, length of stay, laboratory charges, radiology charges, routine charges, and pharmacy charges for patients in each drg, severity, procedure group. subsequently, we compared the resource use of each physician's patients with the norms in the appropriate categories. for each physician , we then accumulated the differences between each of his patient's resource use and the norms, controlling for drg, severity, and procedure type. the are explained in more detail in another article (horn, horn, and moses, 1984). it was found that some physicians treated most of their patients with less resource use than the norms, and some with more. the same comparisons were also made when the patients were grouped only by drg's, and not adjusted for severity or procedure type (figure 8). the two different methods of assessing an individual physician's efficiency often led to different . these disparate signal to a hospital administrator that the underlying causes of the differences should be investigated. only a more detailed review will show whether the differences are because of quality of care, efficiency, or treatment of more severe cases. however, the implications of these differences between drg's and severity- and procedure - adjusted drg's are great. where will medical practice be in the future if the wrong physicians are criticized (or praised) for atypical practice patterns? the in the previous sections have indicated that severity of illness refinements within drg's can produce resource - use groups that are more homogeneous. however, the fact that there is a spread of severity within a drg is not necessarily a fatal problem. if all hospitals treat patient populations with the same distribution of severity of illness within each of the drg's, one might expect variations in resource use within a drg to average out. exactly such a reliance on a law of large numbers was explicitly stated in secretary richard schweiker's report to congress (department of health and human services, 1982) in which he proposed the present prospective payment system for medicare. in figure 9, the distribution of severity of illness within drg's in several different types of hospitals is shown. each hospital the various shaded sub - bars represent the percent of drg's having one severity of illness level (homogeneous with respect to severity of illness), having two severity of illness levels, etc. in hospital ut1, on the other hand, more than 60 percent of the drg's in hospital c1 had one level of severity, and fewer than 10 percent of the drg's had three and four levels of severity. thus, different hospitals have different numbers of drg's with a spread of severity of illness. figure 10 shows the distribution of patients according to the number of severity levels that their respective drg's contained. in hospital ut1, these drg's contained 63 percent of the hospital's patients, compared with 35 percent or less of the patients in the other hospitals in drg's with three or four levels of severity. only 18 percent of the drg's in hospital ut1 were homogeneous with respect to severity, and these drg's contained only 3 percent of the patients in the institution. in the other hospitals, no more than 31 percent of the patients were in drg's that were homogeneous with respect to severity of illness. thus, the drg's that are homogeneous with respect to severity contained only a minority of the patients in the study hospitals, whereas those drg's that were heterogeneous with respect to severity encompassed the majority of patients in all five institutions. however, the distribution of those patients varied greatly by institution. the financial impact of patients in the drg's that were heterogeneous with respect to severity of illness is shown in figure 11. the drg's that contain three or four levels of severity of illness in hospital ut1 (37 percent of all drg 's present in ut1) accounted for 81 percent of the total charges in hospital ut1, compared with 52 percent or less of the charges in the other hospitals. in this respect, thus, different percentages of a hospital's revenue are at risk in the drg's that are heterogeneous. these findings indicate that there can be large inter - hospital differences in the distribution of severity of illness of patients treated within a drg. furthermore, although teaching hospitals frequently treat a greater proportion of more severely ill patients, the severity distributions of the patients in the study hospitals are not predicted reliably by a simple classification by hospital type (teaching versus community) or by number of residents available per bed. the prospective payment system based on drg's is an average - cost - formula system. claims data in the health care financing administration medicare provider analysis and review file supplemented by discharge records from maryland and michigan were used to place patients into their designated drg's, and cost weights per drg were constructed. these weights reflect the relative costs of treating patients in a particular drg across all hospitals. the actual drg payments are computed by multiplying the drg cost weight by standardized amounts with adjustments for region of the country, differences in wage rates, and differences in number of residents per bed. collection of severity of illness data could be accomplished simultaneously with the collection of discharge abstract data on which the drg system is based. professional review organizations, who have been designated to monitor the reliability of discharge abstract data, could also monitor the reliability of severity of illness data. implementation of a prospective payment system involves administrative issues of equity, cost and regulatory burden. an equitable distribution of payments is essential to pay hospitals fairly for the efficient production of services performed. definition of the hospital product as accurately and precisely as possible is of primary importance. unreliable discharge abstract and severity data from coder errors, incomplete records, and potential gaming of the system definitions can be audited and corrected. this problem will exist and will require expense and regulatory efforts to monitor it. to alleviate part of this problem, we are developing a computerized severity of illness information data base that is based on a 6th digit enrichment of the current coding system, the international classification of diseases, ninth revision, clinical modification. this new, richer discharge abstract data set will be the basis for obtaining severity of illness levels from computer algorithms. it will also permit researchers to define and study other case - mix grouping systems from the new discharge data. as mentioned earlier, a patient may become sicker, and hence may receive a higher severity of illness rating, either because of poor quality of care that allows the patient to develop complications, or a poor clinical choice of therapy, to which the patient does not respond promptly. the consequence of this for a prospective payment system would be a higher payment for iatrogenically caused, as well as naturally, caused, illness. this problem exists, however, in the current drg prospective payment system, with or without a severity of illness adjustment. what is needed is a good measure of quality of care to correct this problem in any prospective payment system. a per - case prospective payment system has many of the right incentives to deal with the financial crisis in the health care field. with more accurate descriptions of their products , hospitals should be able to manage themselves in a more businesslike manner, and accurate cross - hospital comparisons can be made. each individual hospital patient is different, but there are sufficient similarities among patients to allow them to be grouped into medically meaningful homogeneous resource - use groups. trade - offs between the number of groups in such a grouping system, their medical meaningfulness, their homogeneity, and the robustness of the system against manipulation by hospitals and physicians need to be investigated thoroughly to determine which of the various possible case - mix systems is most suitable for prospective payment purposes. although our , which used the severity of illness index for case - mix purposes, are consistent within all the hospitals analyses to date, these could be biased by the self - selection factor of the hospitals that have elected to gather severity data. more data from a greater cross - section of hospital types are needed to determine if these will continue to hold for all hospitals.

this article discusses the severity of illness case - mix groups, and suggests a refinement to diagnosis - related groups (drg 's) designed to accommodate the important element of patient severity. an application of the suggested refinement is presented in a discussion of the efficient production of hospital services.the following areas are addressed. a brief summary of the goals and development of the severity of illness index, and the methodology used to collect severity of illness data on hospital inpatients.comparative analyses of the ing case - mix groups within hospitals, and an application of severity - adjusted diagnosis - related groups case - mix definitions.the contribution of the variation in physician practice patterns to the variation in resource use per patient within a hospital.cross-hospital comparisons.some of the consequences of incorporating a patient severity refinement into the prospective payment system.

havana is the capital city of the republic of cuba with 15 municipalities, 2,193,848 inhabitants, and a population density of 3,040/km. located in the north of the country, it covers an area of 720.84 km and has an annual average temperature of 25c. house indexes (percentage of houses with at least one infested container) of 0.05 to 0.91 were reported from 1997 to 2001. in july 2001, house indexes at the municipalities of the city varied from 0.2 to 1.5; however, higher figures were observed at health areas and blocks. these data demonstrate that transmission risk must be assessed in more numerous, smaller geographic areas. the entomologic surveillance and vector control activities involved 4,796 workers; 3,278 family doctors offices (one family doctor per 120 families and 600 inhabitants) and 81 health areas constitute the primary health care system, and 23 hospitals comprise the second and third levels. once the santiago de cuba epidemic was detected in january 1997, specifically in havana city, the surveillance was directed at detecting dengue transmission by studying patients with undifferentiated fever and patients with suspected dengue (patients with fever and two or more symptoms of df such as myalgia, arthralgia, headache, and rash). a serum sample for dengue immunoglobulin (ig) m detection was collected 5 days after onset of fever. igm studies were conducted first at the laboratory of the centro provincial de higiene y epidemiologia de ciudad habana (cphe - ch) by using the ultramicro - enzyme - linked immunosorbent assay (elisa) for dengue igm detection. positive samples were confirmed at the national reference center, the tropical medicine institute (ipk) by an igm capture elisa. a comprehensive study from clinical, epidemiologic, and entomologic perspectives was conducted at those health areas where case - patients were found; a second serum sample was collected 23 weeks after illness onset to demonstrate the antibody seroconversion or a 4-fold increase in antibody titer. the table shows the total number of serum samples studied from 1997 to 2002. cphe - ch, centro provincial de higiene y epidemiologia de ciudad habana; ipk, tropical medicine institute. through march 2002. in june 29, 2001, the index case - patient was a 68-year - old white woman who lived in the 26 de julio health area of the playa municipality; she had no history of travel outside the country. the 26 de julio health area was a residential location with a noncontinous water supply (it received water every 2 days). many persons from dengue - endemic countries lived in the area, and many boarding houses also characterized this area. within 2 weeks, 20 additional df cases were serologically confirmed. a retrospective seroepidemiologic study was conducted in a radius of 1 km around the index patient to look for any patients with suspected dengue or undifferentiated fever; 312 febrile patients, and 14 suspected df patients were found; however, df was confirmed by serologic studies in 4 of them. all 4 case - patients had dengue igm and high titers of igg dengue antibodies. epidemiologic studies of these patients showed that the first case occurred in late may or early june. the primary case - patient was a 53-year - old white man from the same health area as the index patient. once transmission was confirmed, a proactive dengue surveillance program was established, based on information from family doctors. virologic and molecular surveillance demonstrated that dengue 3 was the etiologic agent of the epidemic. ninety - one dengue 3 isolates were obtained from samples collected at various times during the epidemic. considering the active surveillance and that specimens from all identified clinical case - patients were studied by serologic or virologic methods, the figure of confirmed cases is very close to the total number of dengue clinical cases of the epidemic. all confirmed case - patients figure 1 shows the histogram of the epidemic, and figure 2 shows the municipality distribution and the date of confirmed transmission in the city. by week 30 (july) new cases were detected in the arroyo naranjo municipality, and by the end of october (week 42), almost all municipalities had reported dengue transmission. dengue confirmed case notification according to onset of fever extension of the epidemic in havana city, 2001 - 2002 the wide clinical spectrum of dengue was established in the pan american health organization (paho)/world health organization (who) guidelines. because of the detection of dengue transmission in the city, the existence of the primary health system, and the strong dengue surveillance system that included laboratories with appropriate technology for serologic diagnosis, we decided to extend the clinical, epidemiologic, and laboratory surveillance to the study of almost all undifferentiated fever cases and those patients with a compatible classic dengue picture. a house - by - house survey for febrile cases and dengue suspected cases was performed in havana city by the family doctors. as a , 72,162 cases (41,830 undifferentiated fever and 30,332 dengue suspected cases) were epidemiologically, clinically, and serologically studied. dengue infection was confirmed in 12,889 (17.86%) of the total cases. of patients with confirmed cases, 1,660 (12.9%) were children and 11,229 were adults (87.1%); 52.4% were female and 47.6% were male. dhf was diagnosed in 78 patients, all adults (1664 years of age). the main signs and symptoms detected in patients with confirmed dengue cases at the time of hospital admission were fever, 100%; headache, 89%; retrorbital pain, 59.2%; arthralgia, 59.4%; myalgia, 35.2%; and rash, 28.1%. other symptoms such as cough, diarrhea, nausea, and vomiting were observed in 21.2% of case - patients. the peak of the epidemic occurred in october and the highest number of cases occurred on october 20 (241 confirmed cases); 1,150 cases were confirmed by week 42 (october 1420). the onset of symptoms of the last two case - patients occurred by february 22, 2002. the epidemic was considered controlled 36 days later with confirmation that no possibility of transmission existed. case fatality rate was 3.8% among patients with dhf / dengue shock syndrome (dss). after the first cases were detected, all patients with suspected dengue and those who were severely ill, or those classified as having dhf / dss were hospitalized, all adults at the ipk hospital and all children at the aballi and cerro pediatric hospitals. in total, 4,184 patients were hospitalized, 3,197 adults and 987 children. by the end of the epidemic in january 2002, a broad hospitalization policy was established in areas free of vector, aedes aegpyti mosquitoes, (all febrile and dengue suspected case - patients were hospitalized or treated at home with daily visits by the family doctor). the vector control strategy had two phases: the first started as soon as the transmission was detected and restricted the number of cases and geographic extension of the epidemic (the risk of expansion of the epidemic was high because of the vector indexes in havana city and other provinces).the second phase, called the intensive campaign, started at the beginning of january 2002 and interrupted transmission and, consequently, lowered the risk of dengue endemicity in approximately 70 days. the campaign was based on the principles of dengue control established by the paho guidelines with the involvement of the whole community (the head of state, governmental and political bodies at all levels, householders, community organizations, etc .) the objectives of the intensive campaign were to control the vector and interrupt dengue transmission. massive environmental management and sanitation efforts, the elimination of breeding sites, and the elimination of adult mosquitoes were also carried out. these activities were accompanied by extensive efforts to mobilize the community, a strong program of quality control, the active media involvement, and the repositioning of tanks and different water containers. from a house index of 0.49 at the beginning of the intensive campaign, the intensive active surveillance and the hospitalization of all febrile patients and all patients thought to be infected with dengue were crucial in order to reduce the dengue transmission. at present, after 17 months since the last dengue case, strong surveillance is maintained by the six regional laboratories and the national reference center, and no additional cases have been reported. aegpyti in a regional situation in which the disease has caused unprecedented numbers of cases of df and dhf (1,015,420 dengue cases and 14374 dengue hemorrhagic fever, with 225 deaths have been reported to paho) (data provided by jorge arias, who american regional office). as has been stated in the paho resolution approved by the panamerican health assembly in september 2002,

in june 2001, dengue transmission was detected in havana, cuba; 12,889 cases were reported. dengue 3, the etiologic agent of the epidemic, caused the dengue hemorrhagic fever only in adults, with 78 cases and 3 deaths. after intensive vector control efforts, no new cases have been detected.

when the etiology is nontraumatic, there is no pattern to the way in which the joints are impaired, since the consequences of this condition have various effects on radial and midcarpal joints. posttraumatic osteoarthrosis, however, shows a predictable standardized progression, and thus treatment depends on the stage of evolution of this condition. the etiology of posttraumatic wrist osteoarthrosis is usually secondary to ligament injuries or carpal fractures.1 - 5 in posttraumatic wrist arthrosis cases, 95% are located around the scaphoid, and in 55% of the patients with arthrosis the most common pattern is called scapholunate advanced collapse (slac) of the wrist, which from ligament ruptures. the evolution of this type of arthrosis is divided into the following three stages:2,6 arthrosis between the styloid process of the radius and the scaphoid; arthrosis in the radial scaphoid fossa, arthrosis between the capitate and the lunate. the osteoarthrosis can also from pseudoarthrosis of the scaphoid (scaphoid nonunion advanced collapse ( snac)).7 in this degenerative pattern, pseudoarthrosis of the scaphoid acts biomechanically in the same way as injuries to the scapholunate interosseous ligament; as observed in cases of slac wrist, butradial scaphoid fossa was preserved when pseudoarthrosis of the scaphoid occurred.8 the snac stages are: arthrosis between the styloid process of the radius and the scaphoid; arthrosis between the scaphoid and the capitate and arthrosis between the capitate and the lunate. several surgical approaches for the treatment of posttraumatic osteoarthrosis of the carpal bones have been reported namely, proximal row carpectomy, fourcorner fusion, selective denervation of the wrist, partial styloidectomy of the styloid process of the radius, fusion of the scaphoid trapezium trapezoid joint, fusion of the scaphoid capitate joint, atlas fusion (lunate capitate) and the complete fusion of the wrist.4,5,8 - 22 this study aimed to compare the functional of proximal row carpectomy and fourcorner fusion for the treatment of posttraumatic wrist osteoarthrosis with no effect on the midcarpal joint. twentythree patients were selected based on the inclusion and noninclusion criteria for this study (see below). all patients presented wrist osteoarthrosis, with a diagnosis of slac or snac, but without involvement of the midcarpal joint. all patients were operated on by the same surgeon at the same hospital, between august 2004 and september 2007. the inclusion criteria were the presence of wrist osteoarthrosis grade i or ii slac / snac and acceptance of the statement of free and informed consent. the exclusion criteria were presence of gross deformities in other limbs; rheumatological conditions; infections; involvement of the midcarpal joints, diseased condition in the contralateral wrist and previous fractures of the distal extremity of the radius or the carpal bones. the subjective analysis was based on the disabilities of the arm, shoulder or the hand (dash) questionnaire and an analog pain scale.23,24 in the objective evaluation, the following were observed: wrist goniometry, grip force (jamar), grip force of pulp pulp, lateral (key) and threefinger (tripod) pinches, discrimination between two points on the pulp of the second and fifth fingers, and on the dorsum of the first web, measurements of the hand and wrist volumes and the jebsen taylor functional test.25 patients underwent preoperative and postoperative assessments and the latter were done 3, 6 and 12 months after the surgical procedure. all assessments patients who underwent proximal row carpectomy and those who had fourcorner fusion started their rehabilitation with kinesiotherapy and physical means 3 weeks and 2 months after the operation, respectively. comparison of the evolutional within the same surgical group, between the preoperative and postoperative assessment were performed by the wilcoxon test. data on the operated and contralateral wrist were also compared using the wilcoxon test. a nonparametric mann whitney test was used to analyze the between the two surgical groups. the significance level of 5% (p0.05) comparison of the evolutional within the same surgical group, between the preoperative and postoperative assessment were performed by the wilcoxon test. data on the operated and contralateral wrist were also compared using the wilcoxon test. a nonparametric mann whitney test was used to analyze the between the two surgical groups. the significance level of 5% (p0.05) the mean ages of the fusion and the carpectomy groups were 4210.6 and 43.410.1 years, respectively. there was no difference in the handedness of the affected limbs between the two groups. the data were divided into a direct evolutional analysis and comparative analysis within the same group and between the groups. data on direct evolutional analysis direct comparison of evolution of the parameters over time within each group are shown in table 1. after 12 months despite the radial deviation values, all the wrist parameters decreased in both procedures. grip force was evaluated and the fusion group achieved the same values at 12 months as before surgery, this did not occur in the carpectomy group (table 2). for pinch force evaluation, the operated wrists showed volumetry values that were smaller than those of contralateral wrists by the end of the evaluations, after 12 months of surgery, in both groups. the preoperative values in the fusion and carpectomy groups were 7.6 and 8.2, respectively. dash values corresponded to the analog pain scale values, which meant that all patients showed an improvement in their daily living and work activities. preoperative dash values in the fusion and carpectomy groups were 42.7 and 52.4, respectively, and postoperative values were 29.9 and 37.7, respectively. jebsentaylor test values showed that hand abilities had improved in both groups, from 57 to 40.9 seconds in the fusion group and from 74.1 to 65.2 seconds in the carpectomy group. comparative analysis is a proportional comparison of the data between the groups as follows: horizontal analysis: evaluation of the data between the operated and the contralateral wrist 12 months after the operation (table 3). vertical analysis: evaluation of the data on the operated wrist 12 months after the operation in relation to the preoperative ipsilateral data (table 4). in the horizontal analysis, the data were compared proportionally and values for the contralateral limb were used as the baseline. in the vertical analysis, the preoperative measurements were used as the baseline values in the fourcorner fusion group, 1 case of reflex sympathetic dystrophy was seen. in the proximal row carpectomy group, 3 cases of synovitis with significant wrist edema and 2 cases of reflex sympathetic dystrophy were seen. all observed complications occurred no later than the second month after the operation and were treated clinically. none of the patients presented any breakage of the synthesis material or deep infection, or any other condition that might have required further surgical intervention. data on direct evolutional analysis direct comparison of evolution of the parameters over time within each group are shown in table 1. after 12 months despite the radial deviation values, all the wrist parameters decreased in both procedures. grip force was evaluated and the fusion group achieved the same values at 12 months as before surgery, this did not occur in the carpectomy group (table 2). for pinch force evaluation, the operated wrists showed volumetry values that were smaller than those of contralateral wrists by the end of the evaluations, after 12 months of surgery, in both groups. the preoperative values in the fusion and carpectomy groups were 7.6 and 8.2, respectively. dash values corresponded to the analog pain scale values, which meant that all patients showed an improvement in their daily living and work activities. preoperative dash values in the fusion and carpectomy groups were 42.7 and 52.4, respectively, and postoperative values were 29.9 and 37.7, respectively. jebsentaylor test values showed that hand abilities had improved in both groups, from 57 to 40.9 seconds in the fusion group and from 74.1 to 65.2 seconds in the carpectomy group. comparative analysis is a proportional comparison of the data between the groups as follows: horizontal analysis: evaluation of the data between the operated and the contralateral wrist 12 months after the operation (table 3). vertical analysis: evaluation of the data on the operated wrist 12 months after the operation in relation to the preoperative ipsilateral data (table 4). in the horizontal analysis, the data were compared proportionally and values for the contralateral limb were used as the baseline. in the vertical analysis, in the fourcorner fusion group, 1 case of reflex sympathetic dystrophy was seen. in the proximal row carpectomy group, 3 cases of synovitis with significant wrist edema and 2 cases of reflex sympathetic dystrophy were seen. all observed complications occurred no later than the second month after the operation and were treated clinically. none of the patients presented any breakage of the synthesis material or deep infection, or any other condition that might have required further surgical intervention. the mean age (4052 years) of patients in this study is similar to that in other studies.8,16 - 22 a period of 510 years between the trauma and the start of signs and symptoms of arthrosis implies a condition that occurs in patients who are around 35 year old.7 rigor in applying the noninclusion criteria was fundamental for obtaining a homogeneous group of patients. the use of computed tomography to evaluate the joints, particularly the midcarpal joint, contributed greatly towards homogenizing the study groups. twelve months after the operation, the overall range of wrist motion was smaller in relation to the preoperative values in both procedures (p0.05). the arc of flexion extension was, on average, 25% and 17% less in the cases of fourcorner fusion and in proximal row carpectomy, respectively. the radial ulnar deviation, on average, was, 0.3% and 10% less in fusion patients and carpectomy patients, respectively. the radial deviation in cases of fourcorner fusion was the only goniometry parameter that had improved 12 months after the operation. decreases in the overall range of motion in relation to preoperative values in proximal row carpectomy5,26 - 28 and fourcorner fusion5,29 has been reported. in our study, horizontal analysis within each group showed proportional rangeofmotion favoring proximal row carpectomy, except for radial deviation, for which fusion was favored (p 0.05). the presented here are consistent with other published .8,16 - 19,22 however, others found that the arc of flexion extension in cases of fourcorner fusion was greater than the arc in cases of proximal row carpectomy.20 vertical analysis of the fourcorner fusion group showed that the grip force recovered to preoperative values (p0.05). in the carpectomy group, 95% of the preoperative grip force was recovered (p 0.05). a loss of carpal height in the proximal row carpectomy procedure owing to relative stretching of the flexor and extensor tendons has been reported.2 the improvement in grip force after the operation can be credited to pain relief.27,28 horizontal data from a series of proximal row carpectomy cases with longer followup duration have shown that between 60% and 80% of the grip force in the contralateral wrist is achieved.4,26 - 28 in contrast, only 47% of the grip force was achieved in this study. patients undergoing proximal row carpectomy may take up to 1 year to achieve complete rehabilitation.28 horizontal analysis of the grip force in patients undergoing fourcorner fusion provided the best defense of this technique, to the detriment of proximal row carpectomy, because of the preservation of grip force in relation to the nonoperated side.2,16 a 10% increase in relative grip force may occur over a 3year period between postoperative evaluations in cases of fourcorner fusion.30 grip force values of between 70% and 87%, in relation to the contralateral wrist have been reported.8,17 - 20,30 in this study, the grip force was 73% of the contralateral wrist in patients who underwent fourcorner fusion and 47% in patients undergoing proximal row carpectomy compared with the nonoperated side (p0.05). evaluation of the grip force was done by finger pinches (key pinch, pulp pulp pinch and tripod pinch), together with a test to discriminate between two points. this was done to determine whether surgical procedures might have caused lesions in the peripheral nerves.23 the obtained proved that both surgical procedures were safe. the universal analog pain scale was applied to the study patients. in the fourcorner fusion group, there was a 33% reduction in pain, compared with preoperative values (p0.05). in the proximal row carpectomy group the comparative values between groups were not statistically significant. in the vertical dash analysis, the patients who underwent fourcorner fusion or proximal row carpectomy achieved 30% and 28% evolution, respectively, compared with preoperative values (p0.05). in the vertical analysis of the jebsen taylor test,25 the patients who underwent fourcorner fusion achieved, on average, 28% evolution over the task duration, whereas the value was 12% in patients who underwent proximal row carpectomy (p0.05). in the horizontal analysis, almost all patients recovered their function compared with the contralateral side; 98% in fourcorner fusion group and 99% in proximal row carpectomy group. both proximal row carpectomy and fourcorner fusion surgical procedures provided similar functional for treating degenerative conditions of slac / snac without the impairment of the midcarpal joint. indication for the surgical technique should be based on several parameters, such as patient's age, duration of immobilization, risk of pseudoarthrosis, possibility of breakage of the synthetic material, infection, duration of rehabilitation and the experience of the surgical team.

objective: to compare the functional of carpectomy and fourcorner fusion surgical procedures for treating osteoarthrosis following carpal trauma.methods:in this prospective randomized study, 20 patients underwent proximal row carpectomy or fourcorner fusion to treat wrist arthritis and their functional were compared. the midcarpal joint was free of lesions in all patients.:both proximal row carpectomy and fourcorner fusion reduced the pain. all patients had a decreased range of motion after surgery. the differences between groups were not statistically significant.:functional of the two procedures were similar as both reduced pain in patients with scapholunate advanced collapse / scaphoid nonunion advanced collapse (slac / snac) wrist without degenerative changes in the midcarpal joint.

neonatal diabetes mellitus (ndm), defined as persistent hyperglycemia occurring in the first six months of life, is a rare cause of hyperglycemia with an estimated incidence of 1 in 100,000 to 1 in 260,000 live births. almost all cases of ndm have monogenic etiology in contrast to the autoimmune diabetes presenting in children beyond 6 months of age. there are 22 known genetic causes of ndm (mutations in 21 genes and methylation abnormalities at the 6q24 locus) that identify different clinical subtypes of the disease. this includes transient ndm (tndm), permanent ndm (pndm), and complex syndromes in which ndm is often the presenting feature (i.e., wolcott the most common cause of ndm are mutations in the adenosine triphosphate ( atp)-sensitive potassium channel (k - atp channel) subunit genes abcc8 and kcnj11 which regulate the release of insulin from pancreatic cells. the pancreatic -cell k - atp channels are hetero - octamers assembled by four kir6.2 subunits and four high - affinity sulfonylurea receptor 1 (sur1) subunits encoded by the genes kcnj11and abcc8, respectively. kcnj11 mutations are more frequent in patients with pndm, whereas mutations in abcc8 cause tndm more frequently. the majority of infants with ndm are small for gestational age, which may be related to decreased insulin secretion in the fetus and exhibit postnatal catch - up growth with insulin therapy. the most severe defect includes marked developmental delay and early onset epilepsy, also known as dend syndrome. subcutaneous insulin was routinely used in the past to treat patients with this disorder; however, studies have shown that the successful transition from insulin to sulfonylurea (su) agents can be achieved in up to 90% of patients with ndm. we report a case of pndm whose treatment was successfully transitioned from insulin to oral su therapy after 37 years of insulin dependence. a 37-year - old man with history of poorly controlled diabetes with multiple microvascular complications of proliferative diabetic retinopathy, stage 3 chronic kidney disease (ckd) and peripheral neuropathy, presented to our institution for further evaluation. the patient reported frequent hypoglycemia and blood glucose (bg) excursions with bg ranging from 48330 mg / dl. review of his medical records demonstrated previous hemoglobin a1c (hba1c) measures as high as 12.9%. he was diagnosed with type 1 diabetes mellitus (t1 dm) when he was 2 weeks old and multiple daily injections of insulin (mdi) were initiated to control glycemia. he reported a strong family history of early - onset insulin - dependent diabetes in both of his siblings, and his mother had insulin - dependent diabetes mellitus (unknown type). the patient s medical and family history warranted further investigation and consideration of other forms of diabetes. hba1c was 7.7%, c - peptide < 0.2 ng / ml (range 0.83.2 ng / ml) with corresponding bg of 80 mg / dl (65100 mg / dl ; table 1). the patient s physical exam, and specifically the patient s neurological status, was otherwise normal. as genetic testing could not be afforded by the patient, a trial of su therapy was offered. glimepiride 2 mg daily (qd) was initiated and slowly titrated up to 8 mg twice daily (bid) over a 3-month period with an observed increase in c - peptide from undetectable to 0.7 ng / dl (bg 176 mg / dl). glimepiride was then increased to 12 mg bid with further improvement in glycemic control. insulin detemir and insulin lispro were slowly decreased as the dose of su therapy was increased. in an attempt to increase the patient s own endogenous insulin production and achieve further insulin independence, a 7-day trial of sitagliptin 100 mg qd was initiated with a subsequent increase in c - peptide to 0.9 ng / dl (bg 252 mg / dl). however, glycemic control did not improve so the sitagliptin was discontinued and a trial of dapagliflozin 5 mg qd was initiated (despite his ckd stage 3). glycemic control significantly improved, and after dapagliflozin was increased to 10 mg qd, insulin therapy was completely discontinued (his original total daily dose of insulin was 64 units). the patient follows a carbohydrate - controlled diet of approximately 45 g per meal and occasionally takes 3 units of insulin lispro when planning to eat a high carbohydrate containing meal, particularly when eating out at restaurants. the addition of the sodium - glucose cotransporter-2 (sglt2) inhibitor has helped tremendously with postprandial glucose control and the patient has not had any episodes of diabetic ketoacidosis. repeat hba1c after 3 months of oral anti - diabetic therapy was 6.6%, with average bg in the 70130 mg / dl range. over the last year since the initiation of su therapy , his hba1c has remained below 7% without any episodes of hypoglycemia (table 2). multiple attempts to evaluate and test his parents and siblings for ndm have been unsuccessful thus far.table 1c - peptide, glucose, and hba1c values with corresponding insulin and su regimendatec - peptide (0.83.2 ng / ml)glucose (65100 mg / dl)hba1c, % insulin regimen + dose of su7/28/14<0.2807.3det 27 units + lis 7 - 15 - 15 and glim 2 mg qd was added after labs obtained7/30/140.32917/31/14increased glim 4 mg qd8/4/140.4263det 22 units + lis 7 - 15 - 15 + glim 4 mg qd8/12/140.5306changed det to 18 units + lis 7 - 14 - 12 + increased glim to 4 mg bid8/29/140.73219/17/14increased glim to 8 mg bid, decreased lis to 3 - 6 - 59/24/140.7178det 18 units + lis 3 - 6 - 5 + glim 8 mg bid9/25/14decreased lis to 3 units with meals10/6/14stopped mealtime lis, increased glim 12 mg bid10/20/140.71757.0det 15 units + glim 12 mg bid, lis prn, add sitagliptin 100 mg qd10/28/140.9252det 15 units + glim 12 mg bid, stop sitagliptin, start dapagliflozin 5 mg qd2/23/150.62196.6det 10 units + glim 12 mg bid + dapagliflozin 10 mg qd + lis prn, 45 g of cho per meal2/25/15decreased det to 6 units3/4/15glim 12 mg bid and dapagliflozin 10 mg qd, stopped det7/15/151686.9%glim 12 mg bid and dapagliflozin 10 mg qd + lis 3 units prn for high cho meal11/16/151486.8 bid twice daily, cho carbohydrate, det insulin detemir, glim glimepiride, hba1c hemoglobin a1c, lis insulin lispro, prn pro re nata (as needed), qd daily, su sulfonylureatable 2hba1c% trendyear200820092010201120142015hba1c%12.911.711.29.47.76.6 hba1c hemoglobin a1c c - peptide, glucose, and hba1c values with corresponding insulin and su regimen bid twice daily, cho carbohydrate, det insulin detemir, glim glimepiride, hba1c hemoglobin a1c, lis insulin lispro, prn pro re nata (as needed), qd daily, su sulfonylurea hba1c hemoglobin a1c informed consent was obtained from the patient for publication of this case report. ndm is a monogenic form of diabetes that presents within the first six months of life. in approximately half of patients, the diabetes will be permanent, and in the remaining cases the diabetes will remit within a few weeks or months although it might relapse later in life. approximately, two - thirds of tndm cases are caused by abnormalities in an imprinted region on chromosome 6q24 with activating mutations in either of the genes encoding the two subunits of the k - atp channel of the -cell membrane (kcnj11 or abcc8) causing the majority of the remaining cases. in contrast, the genetic abnormality responsible for up to 30% of pndm cases remains unknown although the commonest known cause in outbred populations is mutations in the k - atp channel or ins genes. heterozygous activating mutations in the kcnj11 or abcc8 gene, which encodes the kir6.2 subunits and sur1 regulatory subunits of the k - atp channel, respectively, have been implicated and account for 30% to 58% of cases of pndm diagnosed in patients less than 6 months of age. mutations in the k - atp channels lead to decreased sensitivity to atp inhibition; consequently, the channels remain open in the presence of glucose, thereby reducing insulin secretion. sus close k - atp channels by an atp - independent route thereby causing insulin secretion studies have shown that many patients with diabetes caused by kcnj11 or abcc8 mutations can successfully switch from treatment with insulin to oral su therapy. the dose of su therapy that is required to manage glycemia in patients with ndm, particularly to achieve insulin independence, is often higher than the maximum recommended dose for the treatment of type 2 diabetes mellitus, typically needing around 0.5 mg / kg / day of glibenclamide. in our case, glimepiride was chosen instead of glibenclamide (glyburide) because of the ever - evolving literature reporting a lower risk of hypoglycemia and lower risk of all - cause and cardiovascular - related mortality with the other readily available sus compared to glibenclamide. a report by thurber et al. found that, in patients with ndm, earlier age at initiation of su treatment was associated with an improved response to su therapy. this led the authors to appropriately hypothesize that perhaps the declining sensitivity to su therapy observed with more advanced age may be due to the loss of -cell mass over time in those treated with insulin therapy, thereby highlighting the importance of early recognition and initiation of su therapy. in our patient, we were able to demonstrate a rise in c - peptide as the su dosage was increased. while it is certainly possible that the original c - peptide may have been undetectable because the patient did not follow directions (drink orange juice prior to laboratory assessment to ensure bg is over 200 mg / dl) the observed gradual rise in c - peptide, in the setting of hyperglycemia, as the su dose was increased, demonstrates a slow increase in endogenous insulin secretion in response to the therapy. the patient did follow the directions to raise serum glucose > 200 mg / dl at the time of subsequent c - peptide measures, as demonstrated in table 1. while the lack of genetic testing is a significant limitation of our report, this limitation does not take away from the main point of this report, that being the importance of performing a very thorough personal and family history in patients who present with a prior diagnosis of diabetes, particularly t1 dm very early in life, as a near - miraculous transformation in management may be possible in a minority of patients, as was the case in our patient, even if genetic testing is not available / feasible. genetic testing for some conditions is available free of charge on a research basis in certain academic institutions in patients diagnosed with diabetes before 6 months of age (e.g.,). to the best of our knowledge, we are the first to report the effective and safe use of sglt2 inhibitors in a patient with ndm. while the hba1c did not appear to improve significantly after the addition of dapagliflozin, the therapy did help to lower postprandial glycemic excursions, not adequately addressed by the su therapy, and helped to reduce the frequency at which the patient took a correction dose of bolus insulin, or a dose of bolus insulin to assist with glycemic control when he consumed higher carbohydrate containing meals. we report the case of a patient with long - standing poorly controlled ndm initially misdiagnosed and treated as having t1 dm. many patients with ndm treated with insulin - only therapy behave as brittle diabetics, with difficult to control glycemia leading to complications. the addition or transition to su therapy may help some of these patients achieve better glycemic control, while requiring less dependence on insulin therapy. our case highlights the importance of history taking in the management of patients with diabetes, especially the need to conduct a thorough family history, as recognition of ndm can profoundly impact the diabetes - related management of both the patient and their family members.

neonatal diabetes mellitus (ndm), defined as persistent hyperglycemia occurring in the first months of life, is a rare cause of hyperglycemia and is often misdiagnosed as type 1 diabetes mellitus (t1 dm). numerous reports have shown that the successful transition from insulin to sulfonylurea agents can be achieved in up to 90% of patients with ndm. however, most of the reports pertain to infants; the literature is limited regarding treatment of adults with ndm. we present our experience with a patient with permanent ndm, initially misdiagnosed as t1 dm, who subsequently was successfully transitioned to oral sulfonylurea therapy after 37 years of insulin dependence.

subcutaneous panniculitis - like t - cell lymphoma (sptl) is a rare subgroup of primary cutaneous t - cell lymphoma with major manifestation in the subcutaneous tissues. histologically, sptl is characterized by infiltration of pleomorphic t cells in the subcutis, mimicking lobular panniculitis. given clear differences in disease outcome the world health organization - european organization for research and treatment of cancer (who - eortc) classification for cutaneous lymphomas defines as sptl only cases with an /+ t - cell phenotype and a usually indolent clinical course. in contrast, the aggressive /+ form of cutaneous t - cell lymphomas with frequent subcutaneous involvement was separated as a distinct entity. we describe complete disease remission in a 63-year - old woman with primary cutaneous /-type sptl following treatment with bexarotene and prednisone. in light of a lack of standard treatment for sptl , we describe our experiences with using bexarotene as a first - line therapy and its efficacy in /-type sptl. a 63-year - old woman presented with painful subcutaneous nodules bilaterally on her upper thighs (fig . no other diseases were known . without evidence of pancreatitis, alpha 1-antitrypsin deficiency, or lupus erythematosus, initial treatment with systemic prednisone of up to 100 mg per day and nsaids like ibuprofen led to some improvement without remission . additional histopathology taken 8 months after onset of disease revealed dense lobular lymphocytic infiltrates rimming around adipocytes ( fig . 2a, b) with the infiltrates staining positively for cd3, cd8 (fig . 2c, d), perforin, tia-1, and negatively for cd4, cd5, cd56. complete staging, including ct of thorax and abdomen and head mri revealed no systemic involvement. t - cell receptor polymerase chain reaction (pcr) from skin biopsy was performed by a reference laboratory and revealed clonality as well as an / phenotype. the combination of clinical findings, histopathology and molecular biology prompted us to make the diagnosis of primary / sptl. the patient was started on a treatment of 150 mg / day bexarotene and 80 mg / day prednisone. bexarotene was increased to 300 mg / day while prednisone was tapered in step - wise fashion. complete remission was achieved and bexarotene was discontinued. in the following 2 years two recurrences occurred. for the first recurrence, bexarotene alone was given for 4 months, after which a complete remission was seen. bexarotene alone was again reintroduced for the second recurrence and clinical improvement but no complete remission is currently observed after a follow up of 5 months. sptl was first described in 1991 by gonzalez et al. as a distinct form of cutaneous lymphoma characterized by subcutaneous tissue localization, aggressive clinical course and frequent association with hemophagocytic syndrome. there are two distinct types of sptl with very different disease outcome. in a study of 83 cases of sptl, patients with /+ t - cell phenotype had an indolent clinical course with a five - year survival of 82%. in the same study, patients with /+ t - cell phenotype showed a more aggressive disease with extracutaneous dissemination, and the five - year survival was 11%. this clear difference in clinical outcome necessitates development of separate therapeutic strategies for each type of sptl. bexarotene is a retinoid that selectively activates the retinoid x receptors (rxr). in a clinical trial with heavily pretreated refractory cutaneous t - cell lymphoma (ctcl) patients, bexarotene treatment ed in a response rate of 54% in early - stage ctcl and 45% in advanced - stage ctcl patients. side effects of bexarotene treatment include elevation in lipid and cholesterol levels and hypothyroidism at higher doses. thus, patients on bexarotene need to be closely monitored for toxicity, and lipid - lowering medications and levothyroxine might be necessary. a number of treatment strategies including chemotherapy, systemic corticosteroid therapy, local radiotherapy and surgery have been reported with various success rates. in a study of 63 patients with / sptl, 31 patients were treated with cyclophosphamide, doxorubicin, vincristine, and prednisone (chop) or similar chemotherapy and 19 patients showed complete remission. sixteen of 24 patients went into complete remission after they were given less aggressive initial treatments, including combinations of prednisone, chlorambucil, methotrexate, cyclophosphamide, interferon-?, and gemcitabine we present the first report of complete remission in a sptl patient with combined bexarotene and prednisone treatment. it is not possible to clearly assess the contribution of each substance to the therapeutic effect. but the fact that a prior therapy with prednisone and nsaids did not in complete remission substantiates the contribution of bexarotene. furthermore, after discontinuation of treatment, the patient presented with a relapse of sptl, but went into remission when monotherapy with bexarotene was restarted. in comparison to the dosage used to treat mycosis fungoides , the patient showed remission with relatively low daily doses of 300 mg / day of bexarotene. studies have demonstrated that bexarotene is generally well tolerated at the lower doses (150300 mg / day), which in limited toxicity. low toxicity makes bexarotene a less aggressive and, taking into consideration the indolent nature of sptl, potentially a better initial therapy, as compared to chop or methotrexate. patients with sptl are known to suffer numerous relapses after establishing complete remission. for patients who tolerate bexarotene well , continuous low - dose treatment might be utilized after complete remission to maintain the disease - free state.

subcutaneous panniculitis - like t - cell lymphoma (sptl) of the / type is a rare subtype of non - hodgkin's lymphoma of the skin. although these tumors usually run an indolent course, disease - related morbidity is often severe. clinical findings include subcutaneous tumors located on the extremities or trunk, often accompanied by systemic symptoms like fever or fatigue. due to the low incidence of sptl , no standardized therapy has been defined so far and there is currently no curative therapy available for this type of non - hodgkin's lymphoma. by sharing our experience with bexarotene therapy, we present a safe and potentially improved treatment for patients with sptl. in the case presented, bexarotene was able to induce remission even after recurrence of disease.

cardiac procedures have some of the highest risks of bleeding, with almost 50% of patients experiencing some type of bleeding - related consequence.1 other common surgical procedures have bleeding rates ranging from < 10% to 35%.1 uncontrolled bleeding is associated with increased risk of death,2 blood transfusions, and increased costs due to increased health care resource utilization (hru).3 adjunctive hemostats are intraoperative products that play a critical role in the control of bleeding in the operative setting, and were used in approximately 30% of procedures in 2010.4 there are a variety of adjunctive hemostatic agents that can reduce or stop bleeding. four of the most common are: 1 ) oxidized regenerated celluloses (orcs), 2 ) gelatins, 3 ) flowables, and 4 ) topical thrombins. orcs and gelatins are mechanical hemostats.5 orcs are a plant - derived product (cellulose), typically in a knitted or non - woven fabric form, which work by providing a surface for platelet adhesion and aggregation.6 gelatins are porcine - or bovine - derived products, which typically consist of a dry sponge.6,7 the gelatin products can be moistened with saline,6 in which case they would function like orcs by providing a surface for platelet adhesion and aggregation. however, gelatins are also frequently moistened with thrombin.6 flowables are also gelatin - based, but in the form of a flowable matrix7 that can be applied with a syringe and cannula. thrombins are proteolytic enzymes that aid in the polymerization of the patient s own fibrinogen.5,7 overall, there has been an increase over time in the use of adjunctive hemostatic agents to control bleeding in all surgical procedures.4 the objective of this study was to compare the hru, costs (in us$), and outcomes associated with using orcs compared to other adjunctive hemostats (oahs ; flowables, gelatins, and topical thrombins) for surgical procedures performed in us inpatient settings in which both orcs and oahs were commonly utilized for hemostasis. the hemostatic effect of orcs compared to other hemostat classes has been studied in a number of prospective clinical trials,812 and orcs plus thrombin have been compared to other hemostat classes regarding their impact on inpatient length of stay (los) via a retrospective analysis of us hospital inpatient data.13 however, to the best of our knowledge, the current study is the first to use a cross - sectional dataset to retrospectively analyze the difference in a broad range of hrus, costs, and outcome metrics for orcs compared to other classes of adjunctive hemostats. data for this retrospective, observational cohort study were obtained from the premier healthcare database (referred to as the premier database). premier is a consortium of us community and teaching hospitals that are non - profit and non - governmental, with approximately 6 million discharges annually. the premier database covers approximately 25% of all hospital discharges in the us annually14 and is nationally representative. the database includes patient demographic and hospital characteristics, diagnoses, and procedures, as well as chargemaster data, which includes discharge - level resource utilization. this database contains a day of service - stamped log containing all billed items; laboratory, diagnostic, and therapeutic services rendered; and medications administered during the hospital stay. as an aggregated, de - identified, hipaa compliant and statistically certified database, no institutional review board approval is required for analysis and publication. the premier database was chosen for our current study due to its broad coverage of us inpatient procedure data allowing for generalizability to the entire us hospital market, and the ability to define the patient population and measure outcomes of interest. the study cohort consisted of hospital discharges for adult patients (aged 18 years) undergoing inpatient cardiovascular, carotid endarterectomy, cholecystectomy, or hysterectomy procedures during the 20112012 calendar years, in which adjunctive hemostats in the orc, flowable, gelatin, or topical thrombin classes were utilized; table 1 lists the products included in each class. the only orc products currently approved for use in the us are surgicel (ethicon, inc ., somerville, nj, usa) products flowables, gelatins, and thrombins were combined into a single comparison category, because these products are frequently used in combination during a single procedure. patients with missing data or who were treated with a combination of the orcs and oahs during the same procedure were excluded. cardiovascular procedures, carotid endarterectomy, cholecystectomy, and hysterectomy were selected for evaluation in the present study because both orc and oah usage were relatively common in these procedures, with each of these procedures including at least 3,000 discharges for orc and oah utilization. the original list of procedures considered for inclusion included arteriovenous graft, brain operations, coronary artery bypass graft surgery, carotid endarterectomy, cholecystectomy, and hysterectomy, operations on the kidney, prostatectomy, and valve surgery (figure 1). valve surgery and coronary artery bypass graft surgery were combined to create the cardiovascular procedure group since there was a significant overlap in the patients with these procedures. procedures were identified based on the international classification of diseases, ninth revision, clinical modification (icd-9-cm) procedures and current procedural terminology codes; table s1 lists icd-9 codes. the primary endpoints for this study were los, adjunctive topical hemostatic product units per procedure, total procedure cost, transfusions during hospitalization, and mortality. los and total cost for each hospitalization were abstracted from the dataset as directly supplied by the hospital. the adjunctive topical hemostat product units per procedure abstraction was based on the count of the product units per discharge. standard charge codes for transfused blood products and transfusion procedure codes were used to identify patient discharges with transfusions. all analyses were performed separately for the indications of cardiovascular procedure, carotid endarterectomy, cholecystectomy, and hysterectomy. outcome variables were compared between matched cohorts using standard statistical tests, eg, paired t - tests and mcnemar s tests for continuous and categorical measures, relevant to the outcome variable data type and distribution. to reduce the effect of treatment - selection bias, matched pairs of orc and oah patients were created using a propensity score matching (psm) approach.15 psm is frequently used in the medical literature as a means to adjust for confounding factors when using observational data to estimate the effect of treatments on outcomes.16 once matched cohorts are created, several outcomes can be studied; this method provides an advantage over creating independent regression models for each outcome, because there is less concern about model misspecification.17 in this study, psm was used to create matched patient cohorts based on their propensity to be treated with an orc. to estimate a patient s propensity for a particular treatment, logistic regression models were fit, regressing on patient and hospital characteristics, specifically age, ethnicity, sex, and comorbidities; table s2 identifies codes. pre - existing bleeding conditions, also used to estimate propensity, are listed in table s3. teaching / non - teaching hospital status, hospital census region, and urban / rural hospital location were also considered in the estimation of treatment propensity. additionally, to remove confounding factors due to the effects of other hemostat classes, concomitant use of non - orc / non - oah adjunctive hemostats were matched to isolate differences in outcomes between orc and oah classes. patients were matched using a 1:1 greedy matching algorithm,18 and the balance of all patient and hospital characteristics was assessed. after the matched cohorts were created, differences between orc and oah cohorts were calculated for all outcome measures: los, total costs, transfusions, mortality, and orc / oah units per discharge for each procedure. statistical differences between orcs and oahs for los were assessed with wilcoxon signed - rank sum tests. statistical analysis was performed using sas version 9.2 software (sas institute inc ., cary, nc, usa). data for this retrospective, observational cohort study were obtained from the premier healthcare database (referred to as the premier database). premier is a consortium of us community and teaching hospitals that are non - profit and non - governmental, with approximately 6 million discharges annually. the premier database covers approximately 25% of all hospital discharges in the us annually14 and is nationally representative. the database includes patient demographic and hospital characteristics, diagnoses, and procedures, as well as chargemaster data, which includes discharge - level resource utilization. this database contains a day of service - stamped log containing all billed items; laboratory, diagnostic, and therapeutic services rendered; and medications administered during the hospital stay. as an aggregated, de - identified, hipaa compliant and statistically certified database, no institutional review board approval is required for analysis and publication. the premier database was chosen for our current study due to its broad coverage of us inpatient procedure data allowing for generalizability to the entire us hospital market, and the ability to define the patient population and measure outcomes of interest. the study cohort consisted of hospital discharges for adult patients (aged 18 years) undergoing inpatient cardiovascular, carotid endarterectomy, cholecystectomy, or hysterectomy procedures during the 20112012 calendar years, in which adjunctive hemostats in the orc, flowable, gelatin, or topical thrombin classes were utilized; table 1 lists the products included in each class. the only orc products currently approved for use in the us are surgicel (ethicon, inc ., somerville, nj, usa) products; thus, the dataset only included surgicel - brand orcs. flowables, gelatins, and thrombins were combined into a single comparison category, because these products are frequently used in combination during a single procedure. patients with missing data or who were treated with a combination of the orcs and oahs during the same procedure were excluded. cardiovascular procedures, carotid endarterectomy, cholecystectomy, and hysterectomy were selected for evaluation in the present study because both orc and oah usage were relatively common in these procedures, with each of these procedures including at least 3,000 discharges for orc and oah utilization. the original list of procedures considered for inclusion included arteriovenous graft, brain operations, coronary artery bypass graft surgery, carotid endarterectomy, cholecystectomy, and hysterectomy, operations on the kidney, prostatectomy, and valve surgery (figure 1). valve surgery and coronary artery bypass graft surgery were combined to create the cardiovascular procedure group since there was a significant overlap in the patients with these procedures. procedures were identified based on the international classification of diseases, ninth revision, clinical modification (icd-9-cm) procedures and current procedural terminology codes; table s1 lists icd-9 codes. the primary endpoints for this study were los, adjunctive topical hemostatic product units per procedure, total procedure cost, transfusions during hospitalization, and mortality. los and total cost for each hospitalization were abstracted from the dataset as directly supplied by the hospital. the adjunctive topical hemostat product units per procedure abstraction was based on the count of the product units per discharge. standard charge codes for transfused blood products and transfusion procedure codes were used to identify patient discharges with transfusions. all analyses were performed separately for the indications of cardiovascular procedure, carotid endarterectomy, cholecystectomy, and hysterectomy. outcome variables were compared between matched cohorts using standard statistical tests, eg, paired t - tests and mcnemar s tests for continuous and categorical measures, relevant to the outcome variable data type and distribution. to reduce the effect of treatment - selection bias, matched pairs of orc and oah patients were created using a propensity score matching (psm) approach.15 psm is frequently used in the medical literature as a means to adjust for confounding factors when using observational data to estimate the effect of treatments on outcomes.16 once matched cohorts are created, several outcomes can be studied; this method provides an advantage over creating independent regression models for each outcome, because there is less concern about model misspecification.17 in this study, psm was used to create matched patient cohorts based on their propensity to be treated with an orc. to estimate a patient s propensity for a particular treatment, logistic regression models were fit, regressing on patient and hospital characteristics, specifically age, ethnicity, sex, and comorbidities; table s2 identifies codes. pre - existing bleeding conditions, also used to estimate propensity, are listed in table s3. teaching / non - teaching hospital status, hospital census region, and urban / rural hospital location were also considered in the estimation of treatment propensity. additionally, to remove confounding factors due to the effects of other hemostat classes, concomitant use of non - orc / non - oah adjunctive hemostats were matched to isolate differences in outcomes between orc and oah classes. patients were matched using a 1:1 greedy matching algorithm,18 and the balance of all patient and hospital characteristics was assessed. after the matched cohorts were created, differences between orc and oah cohorts were calculated for all outcome measures: los, total costs, transfusions, mortality, and orc / oah units per discharge for each procedure. statistical differences between orcs and oahs for los were assessed with wilcoxon signed - rank sum tests. statistical analysis was performed using sas version 9.2 software (sas institute inc ., cary, nc, usa). a total of 107,667 patients with a cardiovascular discharge during the 2-year study period were identified in the premier hospital database. after accounting for the inclusion criteria, for the carotid endarterectomy analysis, 30,480 patients were identified during the study period, with 6,689 orc and 9,017 oah patients meeting eligibility criteria. there were 130,931 cholecystectomy patients in the 2-year study period; 12,549 and 3,097 were treated with orcs and oahs, respectively. finally, within the 102,882 identified hysterectomy patients, 5,982 were treated with orcs compared to 7,742 with oahs (table 2). the psm produced cohorts of 22,718 patients for cardiovascular procedures (match rate of 96% relative to orc patients), 10,890 patients for carotid endarterectomy (match rate 81% relative to orcs), 6,090 patients for cholecystectomy (match rate 98% relative to oahs) and 9,348 patients for hysterectomy (match rate 78% relative to orcs), with patients divided equally between orcs and oahs. after matching, patients in the orc and oah groups were similar with respect to demographic data, health status, hospital characteristics, and combinations with other hemostat types (table 2). across all four procedure groups, the number of orc product units used and cost per discharge were considerably lower than for oah (figure 2). carotid endarterectomy gave the greatest differential, with 41% fewer units of orcs used per discharge compared to oahs (p<0.001). average total topical adjunctive hemostat cost was 47% less for orcs than for oahs, equating to a reduction of us$88 per procedure. the other procedures also had considerably lower product units per procedure utilization for orcs compared to oahs (31%, 18%, and 16% lower for cardiovascular procedures, cholecystectomy, and hysterectomy, respectively ; p<0.001 for all three procedure types). differences in mean los between the orc and oah cohorts were generally associated with total discharge costs. in carotid endarterectomy and cholecystectomy procedures, the shorter los for orcs (table 2 ; p<0.001 for both procedure groups) aligned with the lower total procedure costs associated with the orc cohorts in carotid endarterectomy (6% lower ; p<0.001) and cholecystectomy (14% lower ; p<0.001). conversely, in the cardiovascular group, a non - statistically significant 0.2-day longer los for procedures where orcs were used (p=0.948) was observed alongside a 3% higher total procedure cost for orcs (p=0.003). hysterectomy procedures displayed an interesting trend, whereby a longer los associated with orcs (p<0.001) was observed in conjunction with almost equivalent total procedure costs for the orc and oah cohorts (p=0.542). mortality rates for all procedures were low, < 4%, and there was no statistically significant difference between orcs and oahs. a decrease in transfusion rates was found in the carotid endarterectomy and cholecystectomy orc cohort compared to the oah cohort (p<0.001 for both procedures) (table 3). a total of 107,667 patients with a cardiovascular discharge during the 2-year study period were identified in the premier hospital database. after accounting for the inclusion criteria, for the carotid endarterectomy analysis, 30,480 patients were identified during the study period, with 6,689 orc and 9,017 oah patients meeting eligibility criteria. there were 130,931 cholecystectomy patients in the 2-year study period; 12,549 and 3,097 were treated with orcs and oahs, respectively. finally, within the 102,882 identified hysterectomy patients, 5,982 were treated with orcs compared to 7,742 with oahs (table 2). the psm produced cohorts of 22,718 patients for cardiovascular procedures (match rate of 96% relative to orc patients), 10,890 patients for carotid endarterectomy (match rate 81% relative to orcs), 6,090 patients for cholecystectomy (match rate 98% relative to oahs) and 9,348 patients for hysterectomy (match rate 78% relative to orcs), with patients divided equally between orcs and oahs. after matching, patients in the orc and oah groups were similar with respect to demographic data, health status, hospital characteristics, and combinations with other hemostat types (table 2). across all four procedure groups, the number of orc product units used and cost per discharge were considerably lower than for oah (figure 2). carotid endarterectomy gave the greatest differential, with 41% fewer units of orcs used per discharge compared to oahs (p<0.001). average total topical adjunctive hemostat cost was 47% less for orcs than for oahs, equating to a reduction of us$88 per procedure. the other procedures also had considerably lower product units per procedure utilization for orcs compared to oahs (31%, 18%, and 16% lower for cardiovascular procedures, cholecystectomy, and hysterectomy, respectively ; p<0.001 for all three procedure types). differences in mean los between the orc and oah cohorts were generally associated with total discharge costs. in carotid endarterectomy and cholecystectomy procedures, the shorter los for orcs (table 2 ; p<0.001 for both procedure groups) aligned with the lower total procedure costs associated with the orc cohorts in carotid endarterectomy (6% lower ; p<0.001) and cholecystectomy (14% lower ; p<0.001). conversely, in the cardiovascular group, a non - statistically significant 0.2-day longer los for procedures where orcs were used (p=0.948) was observed alongside a 3% higher total procedure cost for orcs (p=0.003). hysterectomy procedures displayed an interesting trend, whereby a longer los associated with orcs (p<0.001) was observed in conjunction with almost equivalent total procedure costs for the orc and oah cohorts (p=0.542). mortality rates for all procedures were low, < 4%, and there was no statistically significant difference between orcs and oahs. a decrease in transfusion rates was found in the carotid endarterectomy and cholecystectomy orc cohort compared to the oah cohort (p<0.001 for both procedures) (table 3). we compared patients treated with surgicel orcs to patients treated with oahs on cost and resource utilization metrics including los, units of hemostat product usage, and transfusions using hospital chargemaster data. after creating comparable cohorts of orc and oah patients with psm, across the four procedures studied (cardiovascular procedure, carotid endarterectomy, cholecystectomy, and hysterectomy), adjunctive hemostatic agent costs were significantly lower when orcs were used instead of oahs. other measures, such as transfusions and readmissions, were similar to or in favor of orcs compared to oahs for most procedure types. los and total procedure cost were lower for orc patients for both the carotid endarterectomy and cholecystectomy procedures. overall, the average cost of orcs was lower than oahs, but the difference in overall procedure costs was greater than the cost difference of the hemostatic agents used. the total cost of treatment was lower for oahs in the procedures in which los was lower. cardiovascular disease was the only procedure in which the los and costs were not lower in orc patients compared to oah patients. it is not possible to discern from the current analysis why cardiovascular disease patients had higher costs when orcs were used, but it may be due to different burdens of bleeding or different requirements for adjunctive hemostat characteristics during cardiovascular procedures, as compared to other procedures. in the present study, cardiovascular patients los ranged from 9.3 to 9.5 days; in a study conducted by stokes et al,3 cardiovascular patients los was similar, with an average of 8.5 days. the stokes study also found that bleeding - related complications or blood product transfusions increased los in cardiac, vascular, and reproductive organ procedures by 4.8, 9.3, and 3.6 days, respectively,3 with cost increases of $ 10,000, $ 15,000, and $ 3,000, respectively. more recently, wright et al found that the frequency of adjunctive hemostatic agent use in major surgeries has increased rapidly over time.4 while studies have been conducted on the reduction of bleeding due to adjunctive hemostat use, or bleeding s impact on costs and resource utilization, there have been a limited number of studies on the impact of adjunct hemostats on both costs and outcomes. a meta - analysis of studies focused on fibrin sealant use did not find a significant difference in the length of hospital stay compared to no adjunctive hemostat use.19 while there have been several studies on the effect of fibrin sealants, there has been very little published on the comparative effects and costs of flowables, thrombins, gelatins, or orcs. a randomized controlled trial evaluating different hemostat options (gelatin, orc, flowable, and thrombin) would be useful to confirm or refute the of the present study. in a previous study of orcs in cardiovascular, carotid endarterectomy, and brain / cerebral operations, the newer, advanced products, surgicel snow (ethicon, inc ., somerville, nj, usa) and surgicel fibrillar (ethicon, inc .), were associated with lower hru and costs compared with surgicel original products.20 the present study found similar outcomes for all orcs compared to oahs. a future study designed to compare the newer advanced products to each other would further help to evaluate adjunctive hemostat options. not all of the factors that influence a physician s choice of products to treat specific patients are represented in the current dataset. additionally, because the patients were not randomized , we can not be certain that there were not inherent differences between the specific procedures or patients receiving orcs and oahs that influenced patient outcomes. however, we attempted to account for this by using psm to create similar groups for comparison. psm can not control for unmeasured characteristics, such as the use of medications prior to hospital admission not captured in a cross - sectional database such as premier, which could influence hemostasis in the operative setting; psm is also less powerful than randomization. however, the patient populations evaluated in the present study were made comparable for all observable characteristics, and were assumed to be similar on unmeasured characteristics such as pre - admission medication use. the data do not capture hospital readmissions to other facilities with different medical record systems and non - hospital - based office visits, and also do not include all outpatient data for all hospitals that submit inpatient data. additionally, it is unclear whether treatment patterns and outcomes differ between hospitals that are within or outside of the premier hospital database and whether these hospitals are representative. however, these are most likely generalizable to many patients across the us, as approximately 25% of all hospital discharges are from a hospital within the premier database.14 outside the us may differ because product availability varies by geography. finally, as with all claims and chargemaster data, coding errors or omitted procedure / product codes could lead to misclassification of patients and potential bias in the . however, since these data are used for payment, there is a strong incentive for accuracy. adjunctive hemostat use has the potential to reduce bleeding complications and associated resource use and costs associated with those complications. as adjunctive hemostat use rises, there is interest in understanding the relative benefits of different hemostat types with respect to clinical and economic outcomes. in the premier database, over 370,000 cardiovascular, carotid endarterectomy, cholecystectomy, and hysterectomy procedures utilized adjunctive hemostats from 2011 to 2012. based on this finding, one may estimate that in the us, for these procedures alone, approximately 750,000 adjunctive hemostat units are used annually in cardiovascular, carotid endarterectomy, hysterectomy, and cholecystectomy procedures. in , while clinical outcomes were similar between procedures where orcs and oahs are used, orc use was associated with fewer adjunctive hemostat units per procedure, as well as lower adjunctive hemostat spending when orcs rather than oahs were utilized. additionally, in carotid endarterectomy, cholecystectomy, and hysterectomy procedures, orc use was associated with lower overall los. in the present study, we also found that procedural adjunctive hemostat costs were at least 50% lower when orcs were used compared to oahs, with similar clinical outcomes. utilizing more orcs compared to oahs during appropriate surgical procedures

adjunctive hemostats are used to assist with the control of intraoperative bleeding. the most common types are flowables, gelatins, thrombins, and oxidized regenerated celluloses (orcs). in the us, surgicel products are the only us food and drug administration - approved orcs.objectiveto compare the outcomes of health care resource utilization (hru) and costs associated with using orcs compared to other adjunctive hemostats (oahs are defined as flowables, gelatins, and topical thrombins) for surgical procedures in the us inpatient setting.patients and methodsa retrospective, us - based cohort study was conducted using hospital inpatient discharges from the 20112012 calendar years in the premier healthcare database. patients with either an orc or an oah who underwent a cardiovascular procedure (valve surgery and/or coronary artery bypass graft surgery), carotid endarterectomy, cholecystectomy, or hysterectomy were included. propensity score matching was used to create comparable groups of orc and oah patients. clinical, economic, and hru outcomes were compared.the propensity score matching created balanced patient cohorts for cardiovascular procedure (22,718 patients), carotid endarterectomy (10,890 patients), cholecystectomy (6,090 patients), and hysterectomy (9,348 patients). in all procedures, hemostatic agent costs were 28%56% lower for orcs, and mean hemostat units per discharge were 16%41% lower for orcs compared to oahs. length of stay and total procedure costs for patients treated with orcs were lower for carotid endarterectomy patients (0.3 days and us$700) and for cholecystectomy patients (1 day and us$3,350) (all p<0.001).costs and hru for patients treated with orcs were lower than or similar to patients treated with oahs. proper selection of the appropriate hemostatic agents has the potential to influence clinical outcomes and treatment costs.

helminths have developed a unique evolutionary dialogue with their hosts' immune system due to their longevity within the host, their complex life cycles, and multicellular nature. these pathogens induce very different immune responses in comparison to bacteria, fungi, viruses, or protozoa. cells of the innate and adaptive immune system are important for the initiation of type 2 immunity, which characterises the response to helminth infection, as well as allergic reactions. the key players in t helper (th) 2-type immunity are cd4 th2 cells and involve the cytokines interleukin (il-)4, il-5, il-9, il-10, and il-13 and immunoglobulin (ig)e. cd4 th2 cells also express some of the cytokines mentioned above as well as the chemokine ligand ccl11 and the chemokine receptor ccr3. these factors lead to recruitment and infiltration of eosinophils, basophils and mast cells, and expansion of alternatively activated macrophages. notably, th2-type immune responses are composed of three major features: inflammation, wound repair, and, most importantly, resistance to helminths. parasites have developed various strategies to modulate the immune system and ultimately suppress host protective th2-type immune responses for example, by induction of innate and adaptive regulatory cells, anti - inflammatory cytokines and specific inhibitory antibody isotypes (reviewed by anthony et al .). hence, helminth parasites are master regulators of immune responses in order to ensure life - long persistence in the host. one strategy of immune regulation that has evolved is the secretion of a wide range of immunoregulatory molecules, which are able to target various host cells and alter them to induce a highly directed host response known as a this response is designed to limit a possibly detrimental th2 immune response, thus restraining the extreme symptoms that are often observed in allergy or in aspects of helminth diseases such as fibrosis in schistosoma mansoni. in immunological terms, the modified th2 response is defined by the development of specific antibody isotypes, including induction of igg4 accompanied by a decrease in ige, as well as il-4 and il-5, while il-10 levels from different regulatory cell sources increase. these mechanisms can lead to attenuation of pathology and clinical symptoms, tolerance, and ultimately persistence of the worm, which is associated with a hyporesponsive immune system. asymptomatic infection assures long - term survival of the parasite within the host and therefore sustains parasite feeding, completion of the life cycle, and successful reproduction. many studies of animal and human helminth infections have shown their potential for downregulating the immune system. moreover, relevant epidemiological studies have observed that helminth - infected populations exhibit lower levels of immunopathological diseases such as th1-related autoimmune diseases or abberrant th2-related diseases for example, asthma or allergic rhinitis. these observations indicate an inverse global distribution of allergy / autoimmune diseases and helminth infections, the first being an expanding problem of developed and industrialized countries, while the latter being a feature in developing countries. such findings support the hypothesis that the immune system has coevolved to operate in the presence of immunomodulatory helminth infections, while in the absence of exposure to helminths, the immunoregulatory components that would normally prevent allergy and autoimmune disease become weakened. here, we focus on recent advances in cellular mechanisms that are employed and modulated during helminth infections as well as on reports from field research and studies on animal models. these studies have identified helminths and helminth - derived products that play a role either in induction of th2 responses and immune modulation in parasitic infections (reviewed in table 1) or in downregulation of bystander th2-type immunopathologies like allergic asthma, allergic inflammation and airway hyperreactivity, food allergy, eczema, atopic dermatitis (th1/th2), or anaphylaxis (reviewed in table 2). it has long been known that helminths induce a specific immune phenotype in the majority of persons that allows for establishment of infection while simultaneously preventing or reducing signs of disease in the host. antigen - specific cellular hyporesponsiveness was described for filarial infections more than 30 years ago by ottesen et al. studying a population in the cook islands endemic for lymphatic filariasis. in this study lymphocytes from adults infected with the filarial species wuchereria bancrofti showed significantly lower levels of proliferation in response to filarial antigen compared with endemic controls who were negative for all signs of infection or disease but constantly exposed to infection and, therefore, putatively consistently exposed to the antigens. later, another study from the same group made the distinction between microfilariae (mf) positive asymptomatically infected persons and mf negative patients showing clinical symptoms of filariasis (e.g., elephantiasis or hydrocoele). the ing data suggested that the outcome of disease depends on the host response together with a mechanism of immune modulation induced by the parasite (reviewed by ottesen). epidemiological studies of helminth - infected persons recognise distinct clinical outcomes that depend on immune regulation induced by the parasite in conjunction with the genetic of the host (reviewed by maizels and yazdanbakhsh). resistant individuals are constantly exposed to the parasite but show no signs of infection or disease; this group develops an appropriate response, defined by equal proportions of th1, th2, and t regulatory (treg) cells with a balance of igg4 and ige levels. a second group develops a hyporesponsive phenotype characterised by asymptomatic infection, which tolerates the presence of fecund adult worms. this group has high levels of regulatory cells and il-10, leading to a modified th2 response. finally, a small proportion of patients develops a hyperresponsive phenotype (characterised by an immunopathological response). in s. mansoni, w. bancrofti, and brugia malayi infections, the main pathological response is a of overreactive t cell responses that cause inflammation and injure the host. this group exhibits increased ige responses, and the treg compartment is greatly diminished. in w. bancrofti and b. malayi infections, this can in elephantiasis, whereby the lymphatic tissue becomes dilated and hypertrophic. parasite death leads to the release of antigenic material that causes lymphatic obstruction in the vessels and chronic inflammation. a second, rare of these infections is tropical pulmonary eosinophilia characterised by chronic lung obstruction, peripheral blood eosinophilia, and extremely elevated levels of ige, greater than in elephantiasis. in onchocerca volvulus infection the rare chronic hyperreactive form, known as sowda, is also defined by high levels of ige. this is accompanied by strong th2 responses, including il-4, il-5, and il-13. thus a fine balance of different aspects of immunity is required to develop a response beneficial to the host. therefore, immune modulation acts in favour of persistent infection and continuous transmission while simultaneously enabling the host to tolerate infection by diminishing clinical symptoms. coinfection studies of helminths with other diseases provide supporting evidence that helminths have great ability to modulate immune responses, some aspects of which may then affect bystander infections. in fact, protective inflammatory immune responses that typically develop in bacterial or intracellular parasitic infections can be downregulated when a coinfecting helminth is present, ing in increased susceptibility in the host to the former infection. for example, individuals coinfected with latent tuberculosis (tb) and w. bancrofti showed lower levels of tb - protective proinflammatory cytokines in vitro than tb patients without filarial infection. research focusing on individuals coinfected with multiple filarial species and plasmodium falciparum demonstrated a modulation of the antimalarial immune response by helminths that increased anti - inflammatory responses. after stimulation with malaria antigen, filarial - infected individuals demonstrated significantly lower levels of interferon (ifn)-, tumour necrosis factor (tnf)- and il-17a, and higher levels of il-10 from cd4 t cells compared with filarial - uninfected individuals. similarly, a study examining the effect of helminth infections on responses to oral cholera vaccine demonstrated that ascaris lumbricoides infection was associated with lower cholera antigen - specific il-2 cytokine responses. these epidemiological studies demonstrate the ability of helminths to modulate immune responses to themselves as well as to concurring and unrelated infections. eradication of helminth infections in industrialised countries in the past 30 years has had a great impact on the prevalence of diseases associated with inappropriate immune responses. the observed increase in the appearance of allergy - related diseases might be a of this and altered hygienic measures in everyday life. one possible explanation is the hygiene hypothesis , which includes a number of factors like improved public health, use of antibiotics and vaccines that in consequence reduce the occurrence of viral, bacterial or helminth infections early in life, contributing to higher numbers of individuals with allergic, and/or autoimmune disorders. there are numerous cohort studies determining allergic disease status and immune responses to parasite - specific antigens or environmental allergens of helminth - infected individuals in parasite - endemic areas (reviewed by others). however, when interpreting this data it is important to take into consideration the time of first infection, duration and intensity of infection, helminth species, as well as host genetic and environmental factors. infections with trematodes, whipworms and hookworms were described to be negatively correlated with the allergen skin prick test. from a study of gabonese school children tested for skin reaction to house dust mites (hdms) and other allergens, for schistosoma eggs in urine and microfilariae of loa loa and mansonella perstans in blood, showed lower prevalence of a positive skin test to hdms in children with urinary schistosomiasis. moreover, schistosome antigen - specific concentrations of il-10 were significantly higher in infected children and concentrations of il-10 were negatively associated with a positive skin test . several population studies done in endemic areas of schistosoma infections in brazil and of ascaris, trichuris, and ancylostoma in ecuador revealed a strong inverse association between helminth infections and immediate skin test reactivity to common environmental aeroallergens. cooper et al. reported a negative association between very high levels of ige antibodies and allergen skin test reactivity. it is possible that anti - helminth - ige competes for the same high - affinity ige receptors targeted by allergen - specific ige. although other studies from africa reported on a reverse association between hookworm infections and sensitization to hdm dermatophagoides pteronissinus (der p 1), they did not show a significant association between skin test sensitivity to hdms, allergen - specific iges and between total serum ige levels and helminth - specific iges. the contribution of helminth infections to the inverse correlation with allergy - related diseases was demonstrated in studies with anthelminthic treatment. from a randomized, controlled trial on the effect of repeated treatment with anthelminthics on a population of gabonese school children showed a significant increase in the rate of developing skin sensitivity to hdms. this was in part mediated by reductions of ascaris and/or trichuris infections, directly pointing to the importance of a presence of live worms to suppress allergic responses during infection and also preventing allergy later in life. lynch et al. also reported an effect of anthelminthic treatment on the allergic reactivity of children in the tropics in venezuela. the authors showed that the effective elimination of intestinal helminth infection ed in significantly decreased total serum ige levels. however, immediate - hypersensitivity skin test reactivity and serum levels of specific ige antibody against common allergens were significantly increased in treated children. also other studies reported that anthelminthic treatments increase allergen skin reactivity to environmental allergens. rodrigues et al. reported that brazilian children with heavy infection in early childhood with trichuris trichiura had a lower prevalence of allergen skin test reactivity to a wide spectrum of common environmental allergens (hdms, blomia tropicalis, fungi, animal, and cockroach allergens) later in childhood, even when the helminth infection was no longer present. additionally, a population study in taiwan showed that schoolchildren bearing pinworm enterobius were diagnosed with lower prevalence of asthma and allergic rhinitis; moreover, these children had reported an infection of pinworms early on in life. thus, early exposures and infections with helminths may have a protective effect and suppress allergic inflammation later during life. additionally, host genetic factors may play a role in prevalence of helminth infections and allergies. notably variants of genes involved in th2 immune responses like il13 or stat6 were associated with increased risk of asthma and allergy. studies among african - americans shed a light on the limited understanding we have on asthma genetics. polymorphisms in candidate genes associated with asthma showed higher frequency among certain ethnic s that developed strong th2 immune responses. thus, genes, which in the evolution of the immune system were initially responsible for induction of th2-type immune responses and for parasite expulsion, now cause problems with allergy - related diseases when living in sterile and modern societies. in contrast to population studies reporting a negative correlation between allergy and helminth infections as discussed above, various studies from south america, europe, and asia on a. lumbricoides infections and allergy reported increased rather than decreased prevalence of asthma. for example, two cross - sectional surveys among schoolchildren in former east germany revealed that children with low doses of helminthic antigens (low worm burden) but ascaris - ige seropositivity had higher levels of total ige and higher prevalence of allergic rhinitis and asthma to inhalant allergens. it was proposed that heavy parasitic infections might generate immune suppressive mechanisms, whereas mild worm exposure and low - level contact with helminths may enhance reactivity to environmental allergens or even potentiate the th2 immune response that in some cases may promote allergic inflammation. as ascaris infection has been well - documented as a risk factor for asthma (described elsewhere), there are two main hypotheses why ascaris promotes allergic reactions. the first relates to the specific pulmonary phase in the ascaris life cycle that causes inflammation and eosinophilia, which in ige - mediated asthma with high total- and specific - ige titres against larvae and adult worms. the second hypothesis of increased allergy in ascaris infection is explained by cross - reactivity of ascaris - specific molecules with environmental allergens like hdms or the storage mites b. tropicalis that induce allergen - specific iges leading to strong th2-type responses and therefore contributing to overall pathogenesis. moreover, ascaris - tropomyosin, a pan allergen, induced wheal and flare in skin prick tests and histamine release from basophils of asthmatics and nonasthmatics. these suggest that ascaris - tropomyosin in some allergic individuals potentiates the pathogenesis of asthma and other allergic diseases. interestingly, the promotion of atopy can also be observed in parasitic infections where the human is not the definitive host and, therefore, chronic infection is not established, for example, toxocara spp.. in addition, the nematode anisakis simplex, regularly infecting marine mammals as definitive hosts but accidentally also leading to gastrointestinal (gi) infections in humans, who ingest raw fish, was described to cause allergic reactions. allergy to a. simplex is being recognised as an occupational disease with a wide spectrum of allergic reactions (rhinitis, asthma urticaria, allergic contact dermatitis, and anaphylactic shock). as both parasites cause accidental and infrequent infections, in case of infection the immune system may be exposed to th2-inducing events rather than to immunomodulatory mechanisms that occur in chronic infections. it is important to keep in mind that variations in epidemiological studies may have many confounding factors like types of parasitic infections, intensity and timing of the infection, other coinfections, first exposure to allergen as compared to parasitic infection, types of allergens, nutritional status of the patients, medication received, and genetic . current research has focused particularly on identifying the cells targeted by parasitic immune modulation, and the exact helminth - derived molecules responsible for this. immune modulation by the parasite occurs through production of specific parasite - derived molecules that target mammalian host immune cells and signaling pathways. during infection this is strictly dependent on live parasites as shown by the recovery of cellular hyporesponsiveness in patients treated with anthelminthic chemotherapy. in addition, a study by da'dara and harn demonstrated that killing of schistosome parasites could reinstate immune responses to a bystander vaccine targeting human immunodeficiency virus-1, which were greatly diminished in the presence of the parasite. similarly, another study demonstrated that prior elimination of intestinal helminths in patients vaccinated with bacille calmette - guerin ed in greater protective responses to the vaccine compared with patients who did not receive anthelminthic chemotherapy. during active infection, both gi and tissue - dwelling helminths can establish long - term chronic infections and have many immunomodulatory molecules in common, which, despite the different anatomical locations target similar host cells employing comparable regulatory mechanisms. thus, experimental models together with human studies have helped to elucidate the mechanisms and cell targets underlying parasite - induced immune modulation. in this section, presenting cells (apcs) required in priming the adaptive immune response, dendritic cells (dcs) are an essential driving force of th2 induction in helminth infections and in allergy - related diseases. the field of research on dcs targeted by parasite - induced immune modulation is as vast as that on macrophages, and there is definitive evidence of a specific dc phenotype that develops in helminth infection, demonstrating modulation by the parasite to deviate host inflammatory responses. several groups could show that in helminth infection, dcs mature only selectively or show impaired functions in terms of tlr responsiveness. as dcs are the main messenger cells to communicate with t cells and initiate an immune response, interference with their functions represents a key mechanism for helminths to induce an environment conducive to their survival. the downregulation of proinflammatory cytokines appears to be a frequent feature in helminth - mediated modulation of the th2-type response. murine dcs stimulated with lipids from s. mansoni eggs matured to induce specifically tregs that produced il-10, reducing th1 responses whilst producing a modulated th2 response. dcs that have been previously exposed to helminth products can also effectively prime th2 cells. s. mansoni soluble egg antigen (sea) is a particularly strong inducer of th2 responses, and importantly the glycoprotein omega-1 was identified as the main th2-inducing component in sea. human dcs exposed to b. malayi mf showed higher levels of apoptosis and decreased production of il-12 and il-10. in fact when human monocytes that were being differentiated to dcs in vitro were stimulated with b. malayi mf antigen, they produced significantly decreased levels of il-12p40, il-12p70, and il-10 in response to bacterial adjuvant. massacand et al. also demonstrated how dcs exposed to excretory - secretory (es) products from both nippostrongylus brasiliensis and heligmosomoides polygyrus had reduced expression of the proinflammatory cytokine il-12/23p40. similarly, balic et al. reported that bone - marrow - derived dcs exposed to n. brasiliensis es (nes) products actively matured and could induce a th2 response when transferred into nave mice. high levels of il-4, il-5, and il-10 were detected after reexposure to nes. in vitro, these dcs produced high levels of il-6 and il-12p40 but full production of il-12p70 was blocked. finally it was shown that murine lipopolysaccharide- (lps-) or cpg - matured dcs exposed to es products from taenia crassiceps showed diminished responses to stimulation through tlrs 4 and 9. these dcs significantly decreased levels of proinflammatory cytokines, including il-12p40 and il-12p70, as well as tnf- and il-15. thus, downregulation of proinflammatory cytokines seems to be a frequent mechanism in immune modulation by helminths. combined with suppression of proinflammatory cytokines, a key aspect in modulation of dcs is the downregulation of costimulatory molecules, leading to induction of a th2 response.. illustrated that stimulation of murine bone - marrow - derived dcs with a. lumbricoides pseudocoelomic body fluid (abf) influenced dc maturation by inducing expression of il-6, il-12p40, and macrophage inflammatory protein 2. however, dc maturation was only partial as levels of costimulatory molecules cd80, cd86, cd40, ox40l, and major histocompatibility complex (mhc) class ii were not affected. furthermore, when dcs were cultured in the presence of ovalbumin (ova) with abf and then transferred into nave mice, restimulation ed in significantly increased levels of il-4, il-5, and il-10 compared to dcs cultured without abf. coculturing of es products but not adult worm antigen from h. polygyrus with lps - treated dcs induced a semimature phenotype with moderate expression of cd40, cd86, and mhc class ii. in fact when dcs were treated with es and ova and subsequently cultured with ova - specific t cells, the t cells developed a regulatory phenotype, expressing cd25 and high levels of il-10. furthermore, a novel subset of dcs expressing low levels of cd11c have also recently been identified in mice chronically infected with h. polygyrus. in this infection setting, dcs acted as efficient apcs and induced high numbers of cd4 t cells expressing foxp3 but only low numbers of cd4 effector t cells. this population of dcs was suggested to be one of the main target cells involved in inducing an immunoregulatory environment in h. polygyrus infection, allowing the establishment of chronic infection. nevertheless, dcs are clearly essential for efficient priming of the th2 response, as depletion of cd11c cells during development of the adaptive cd4 t cell response in s. mansoni infection drastically impaired the th2 response. macrophages that are activated by the th2-type cytokines il-4 and il-13 develop an alternatively activated phenotype and have a well - described role in helminth infections (reviewed by hoerauf et al .). alternatively activated macrophages (aams) are recruited in large numbers to the sites of helminth infection where they can proliferate, as recently shown. aams are characterised in mice by expression of specific markers, including arginase-1, resistin - like molecule (relm)- (also known as found in inflammatory zone, fizz1), ym-1, ym-2, acidic mammalian chitinase (amcase), and mannose receptor c type (mrc)-1. aams are important in tissue homeostasis, downregulation of the adaptive immune system, acting as effector cells against parasites, and to reduce or heal any ensuing damage caused by infection. aams recruited during b. malayi infection have been demonstrated to drive cd4 th2 responses, deviating the immune system from inducing a proinflammatory th1 response that could be detrimental to parasite survival. monocytes from b. malayi asymptomatically infected patients have also been described to show an alternatively activated phenotype, expressing the aam - specific markers arginase-1, mrc-1, resistin, and ccl18 as well as the downregulatory cytokines transforming growth factor (tgf)- and il-10 (similar to what has been described for mouse aams). however, translation of characterised markers into the human system in general should be done with caution, as reports indicate arginase-1 may not be a reliable marker for aams in humans as it is found in other cell types. furthermore, human monocytes do not express arginase-1 after stimulation with il-4 and il-13, unlike mouse macrophages (reviewed by raes et al .). this indicates that the translation from murine to human data can be difficult, and reliable markers for alternative activation in human monocytes should be further investigated. macrophages from b. malayi infected mice were shown to be fully capable of processing and presenting antigen and providing costimulation for t cells, however the ing antigen - specific t cell population remained suboptimal with cell proliferation but not th2 cell cytokine production being impaired. these macrophages were il-4-dependent and required direct contact with t cells to induce hyporesponsiveness. in a mouse model of s. mansoni infection, arginase - expressing macrophages played an essential role in host protection by suppressing expression of the classical inflammatory cytokines il-12 and il-23. removal of these cells ed in an accumulation of parasite eggs in the intestines and intestinal haemorrhage, thus preventing the eggs from being excreted. large numbers of aams were shown to accumulate in n. brasiliensis infection in the gut, characterised by expression of arginase-1, relm- and ym-1, in an il-4- and il-13-dependent manner. macrophage depletion ed in impaired worm expulsion from the small intestine and prevented smooth muscle hypercontractility. relm- was shown to be essential in regulating lung inflammation, granuloma size and fibrosis in mice challenged with s. mansoni eggs. moreover, pesce et al. illustrated that challenge of relm- deficient mice with s. mansoni eggs ed in elevated eosinophil levels and ige titres. in the same study relm- deficient mice challenged with n. brasiliensis infection produced increased th2 responses, manifested by elevated lung pathology and expulsion of adult worms. importantly, addition of exogenous relm- reversed the pathological th2 responses in this infection. this represents relm- as a key host - derived molecule induced by helminth infection to allow prolonged parasite survival. these studies point at a host protective role for aams in specific tissue dwelling and gi helminth infections. among the cell types targeted during helminth infections, natural cd4 tregs develop in the thymus and express the transcription factor foxp3, the il-2 receptor chain (cd25), ctla-4, and a range of immunosuppressive proteins including il-10 and tgf-. additionally, other adaptive cd25 treg populations exist in the periphery that develop from nonregulatory t cell subsets, with their expression of il-10 and tgf- distinguishing them from activated cd4cd25 nonregulatory t cells. tregs are important in reducing pathology in the host via suppression of both th1 and th2 responses, thus preventing disease symptoms as demonstrated by evidence of treg involvement in both th2-mediated allergies and th1-mediated autoimmunity (reviewed by else). in a murine model of schistosomiasis cd4cd25 t cells expressing high quantities of il-10 were shown to play a significant role in reducing immunopathology, especially in the chronic stage of infection. mckee and pearce illustrated that in s. mansoni - infected mice cd4cd25foxp3 cells produced significant levels of il-10 that were required to prevent dc - derived il-12, thereby suppressing th1 responses. in fact, isolated schistosome eggs were demonstrated to be capable of modulating th2 responses, without the need for the former life cycle stages that develop during infection. schistosome eggs injected into mice were shown to induce a population of foxp3 cells that expanded to control the cd4 t cell response, preventing inflammatory th1 responses while modulating th2 responses to prevent th2-mediated immunopathology. both natural and adaptive tregs have been described in filarial - infected persons, with the adaptive treg population producing high levels of il-10. our group has shown that in h. polygyrus infection, foxp3 tregs are a requirement for limiting immunopathology and represent a potential source of il-10. thus, while treg depletion did not affect worm burdens, the th2 response was greatly accelerated and augmented, with high levels of il-4 and il-13 being produced. the uncontrolled th2 response led to increased immunopathology in the intestine, demonstrating that tregs were essential in regulating this response. foxp3 tregs were also induced after stimulation with h. polygyrus es products, reducing proliferation of effector cells by activating the tgf- signaling pathway. during infection, tregs may therefore be seen as important effector cells required to prevent or reduce pathology in the host by modulating the ensuing th2 response, thereby simultaneously allowing establishment of chronic infection. host protection as well as regulation by antibodies and b cells is being recognised as an essential component in th2 responses in helminth infections. in s. mansoni - infected mice, where the dominant isotypes are igg1 and ige, blockade of b cell production ed in high levels of the proinflammatory cytokines ifn- and il-12 but low levels of the th2 cytokines il-4 and il-10 in acute infection. moreover mice deficient in b cells could not downregulate granuloma formation in the chronic stage of infection, and this mechanism was mediated by fcr, indicating a role for antibodies in down modulation of pathology. in fact, antibody isotypes are demonstrated to have an important role in determining the outcome of helminth infection in the host. the cytokines il-4 and il-13 act on b cells to induce both igg1 and ige in mice and igg4 and ige in humans. high levels of igg4 but low levels of ige are found in the blood of hyporesponsive, asymptomatic persons infected with b. malayi, w. bancrofti, and o. volvulus. igg4 correlates with high levels of il-10 and the presence of adult worms in hyporesponsive persons. in bancroftian filariasis , high levels of igg4 but low levels of ige were found in mf positive individuals compared to patients with clinical disease (elephantiasis or tropical pulmonary eosinophilia). ige is known to activate degranulation of mast cells (mcs), basophils and eosinophils and induce antibody - dependent cell - mediated cytoxicity (adcc). igg1 in mice and igg4 in humans compete with ige for binding sites and therefore may inhibit such processes. thus, inhibitory igg4 may prevent immunopathological responses in helminth asymptomatically infected individuals and can simultaneously provide an indication of the clinical outcome in infected persons. regulatory b cells have been first described in autoimmune diseases, where the main mediator of suppression is il-10. helminth infections can also induce specific b cell phenotypes with regulatory properties as shown in infection with s. mansoni and h. polygyrus. in s. mansoni infection, a particular subset of b cells has been described that are cd1d and express high levels of il-10 (defined as cd19il-10cd1dcd5cd21cd23igdigmcells). transfer of these cells into ova - sensitized mice reduced ova - induced allergic airway inflammation via induction of foxp3 tregs; the effect was tgf- independent as showed in anti - tgf- mab treatment experiments. furthermore, another group demonstrated that transfer of il-10-producing b cells from il-4 deficient mice infected with s. mansoni provided complete resistance to experimentally induced anaphylaxis when applied to nave mice. thus, absence of il-4 was essential to prevent exacerbation of this allergic response. in another study, the same group proved that the protective response of s. mansoni infection was entirely dependent on b cells as depletion of igm b cells ed in mice becoming completely susceptible to anaphylaxis. interestingly, adoptive transfer of a population of cd19cd23 b cells from h. polygyrus - infected mice could reduce the effects of allergic airway inflammation of ova - sensitized mice, however, in an il-10 independent manner. these mesenteric lymph node b cells reduced secretion of il-5 and infiltration of eosinophils into the airways, suppressing allergen - induced pathology. thus, b cells from helminth infections exhibit regulatory capacity in unrelated diseases and are of therapeutic interest in allergies, therefore their phenotype and functional aspects should be further investigated. there are numerous studies examining the effects of different helminth infections (e.g., s. mansoni , h. polygyrus , n. brasiliensis , trichinella spiralis , and litomosoides sigmodontis) on allergy - related diseases in experimental animal models, where helminths show the capacity to suppress abberrant th2 immune responses. in addition, es products either from h. polygyrus or n. brasiliensis were described to have similar beneficial immunoregulatory effects. furthermore, helminths act through various cell types and can interfere with allergy symptoms in animal models via distinct mechanisms including tregs , b cells , and induction of regulatory cytokines like il-10 or tgf- , as discussed in the previous section and reviewed in table 2. these and other studies led to the discovery of specific immunomodulatory helminth - derived molecules and products that induce a microenvironment beneficial to the parasite, while at the same time preventing immune - related pathology associated with vigorous th2 responses (figure 1). however, as mentioned earlier, persistence of the parasite is not obligatory to reduce allergic symptoms in later life. moreover, these functionally and structurally diverse molecules are expressed throughout the parasite life cycle and interact directly with host cells. a summary of helminth - derived immunomodulatory molecules and products is given in table 1. we focus here on molecules that are relevant for human disease, established animal models of host - parasite interactions, and defined molecules that downregulate unrelated th2 inflammation. the existence of parasite - derived homologues of host mammalian anti - inflammatory cytokines reveals an apparent evolutionary struggle between the parasite and the host, whereby the parasite has evolved mechanisms to establish chronic infection. for example, two tgf- homologues found in brugia species, bm - tgh-1 and bm - tgh-2, have been well characterised, the second of which is thought to have an immunomodulatory role. tgh-2 is secreted by adult worms and in its recombinant form was shown to bind the human tgf- receptor, thus potentially influencing treg development. s. mansoni male worms express a member of the tgf- receptor family known as smrk-1 for which mammalian tgf- may be a ligand involved in worm development. h. polygyrus es contains remarkable tgf--like activity, inducing foxp3 expression in nave t cells and modulating immune functions, thereby maintaining worm burdens to induce chronic infection. the beneficial role of homologues of mammalian chemokines such as macrophage migration inhibitory factor (mif) is less apparent. mif homologues have been described in multiple helminth species (reviewed elsewhere). helminth mif has direct chemotactic effects on human monocytes but appears to be associated with anti - inflammatory, modified th2-type responses. in a murine model of asthma, a recombinant mif - like protein from a. simplex (as - mif) was described to have a protective effect and to suppress the th2-type response, reduce production of il-4, -5 and -13 in the bronchoalveolar lavage fluid (bal), inhibit eosinophilia, goblet cell hyperplasia, and ameliorate lung hyperreactivity. the effect of as - mif was possibly mediated by il-10 and tgf- production in the bal as well as via treg induction in the lungs of as - mif - treated animals when compared to asthmatic controls. another helminth - derived molecule with structural similarities to mammalian chemokines is ancylostoma - secreted protein-2 (na - asp-2) secreted by the infective larvae of necator americanus hookworm. recombinant na - asp-2 was shown to recruit high numbers of neutrophils when injected into an air pouch (a sterile inflammatory setting) in mice. the influx of neutrophils was suggested to create permeability in host vessels, thus facilitating larval migration. hence, cytokine homologues seem to be exploited by the parasite for efficient immune evasion; however more work is needed to assess which of those molecules participate in suppression of unrelated inflammation. the cystatins and serpins are the best - characterised protease inhibitors of helminths that have immunomodulatory potential. mammalian cysteine proteases are essential for efficient processing and presentation of antigen on mhc class ii to induce an appropriate adaptive t cell response. mammalian cystatins play a vital role in regulating these pathways; however, helminth cystatins from acanthocheilonema viteae, b. malayi, n. brasiliensis, and o. volvulus have been shown to interfere with this process to dampen antigen - dependent immune reactions (reviewed by klotz et al .). bm - cpi-2, a cystatin from b. malayi, was illustrated to interfere with antigen processing, which led to a reduced number of epitopes presented to t cells in vitro. studies from our own group have demonstrated that onchocystatin (rov17) from o. volvulus reduced antigen - driven proliferation of peripheral blood mononuclear cells in a monocyte - dependent manner. recombinant avcystatin (rav17) from a. viteae has potent immunomodulatory roles illustrated by its ability to reduce antigen - specific and unspecific t cell responses. avcystatin is recognised by macrophages and upon uptake induces phosphorylation of the mitogen - activated protein kinase signaling pathways erk1/2 and p38 in macrophages. this led to tyrosine kinase - dependent il-10 production in macrophages. furthermore, we could show in an ova - induced airway hyperreactivity mouse model that avcystatin - treated asthmatic mice exhibited amelioration of the disease. avcystatin administered intraperitoneally (i.p .) during the sensitization phase as well as before challenge with ova, suppressed recruitment of eosinophils into the lungs, ova - specific and total ige levels, and reduced allergen - specific il-4 production. il-10 is a key element in avcystatin - induced immunomodulation, as blocking of il-10 with an anti - il-10r mab reversed the beneficial effect on cell recruitment and production of ige. moreover, depletion of macrophages with clodronate liposomes before airway allergen challenge diminished the antiallergic effect of avcystatin. the rodent filarial species l. sigmodontis secretes a cystatin at various stages of the life cycle, which after injection via microosmotic pumps into the peritoneal cavity of l. sigmodontis - infected mice greatly decreased nitric oxide production and proliferation of antigen - specific spleen cells. similarly, recombinant cystatin from the es product of the gi nematode n. brasiliensis (named nippocystatin, nbcys) was shown to inhibit cathepsins l and b, and suppressed antigen processing by apcs. furthermore, ova - sensitized and nbcys - treated mice showed decreased ova - specific spleen cell proliferation, reduced ova - specific ige levels and cytokine production due to inhibition of cathepsin b and l - dependent antigen processing. similar to cystatins, serpins (serine protease inhibitors) have important roles in mammalian biological processes including regulation of complement activation, inflammatory pathways, and cell interactions. bm - spn-2 is a serpin expressed by b. malayi microfilariae, which could inhibit proteases of human neutrophils, thereby interfering with and potentially circumventing the most abundant leukocyte to encounter mf in the bloodstream. however, another study by stanley and stein contests the enzymatic activity of bm - spn-2. taken together, protease inhibitors such as cystatins are a class of molecules found in numerous helminths that have important immunomodulatory functions. a secreted 62 kda glycoprotein from a. viteae known as es-62 is one of the best - characterised helminth immunomodulators. es-62 has been shown to exhibit a plethora of well - documented anti - inflammatory properties and contains phosphorylcholine moieties, which are largely responsible for immunomodulation (reviewed by harnett et al .). ova - specific cd4 t cells exhibited lower levels of proliferation and il-2 production after challenge with es-62, as well as inhibition of il-4 and il-13. it was also shown that es-62 acts on macrophages to inhibit production of il-12 if the cells were subsequently exposed to lps and ifn-. es-62 could also be shown to act on bone - marrow - derived precursors of dcs to inhibit a proinflammatory response induced by lps and b cells by modulating t and b cell interactions. it directly inhibited fcri - induced release of allergy mediators from human mcs by selectively blocking key signal transduction events. es-62 also interfered with mcs in vivo in a mouse model of immediate - type hypersensitivity to oxazolone, thereby diminishing ear swelling and mc - dependent hypersensitivity, as well as ex vivo mc degranulation in mice that were previously injected with the molecule. moreover, es-62 ameliorated ova - induced murine airway inflammation, airway hyperresponsiveness, lung pathology, and eosinophilia by suppression of mc function. thus, es-62 is likely to be a key mediator of filarial - induced immunoregulation, and its properties might be used to dampen overwhelming unrelated inflammation in the future. schistosome soluble egg antigen (sea) and es products released by the egg stage of the parasite contain potent th2-inducing and immunomodulatory activity. sea from s. mansoni was shown to be an extremely strong inducer of th2 responses without the need for live infection or the addition of adjuvant. recently it was shown that omega-1, a hepatotoxic ribonuclease, is one of the key players in this response. omega-1 is a glycoprotein, which was demonstrated to polarise human monocyte - derived and cd11c murine dcs in a direction supporting th2 responses even in the presence of lps. in fact, sea depleted of omega-1 was not able to sufficiently induce a th2 response in vitro. however, the th2-suppressive actions of schistosome - derived antigens clearly highlight the ability of this helminth to modulate host immune responses. es- or omega-1-treated dcs display the typical modulated phenotype that is critical for induction of a th2 response, including reduced expression of costimulatory molecules and a lowered efficiency in participating in dc - t cell conjugates. in fact, this study demonstrated that omega-1 was shown to alter the morphology of dcs, possibly preventing t cell activation. ipse / alpha-1 is another glycoprotein present in sea and abundant in es, which could induce il-4 production from human basophils in an ige - dependent but antigen - independent mechanism. demonstrated that glycan - containing sea could induce high levels of il-5, ige production, and eosinophilia when administered intranasally into mice. the same group was subsequently able to show that s. mansoni sea contained the carbohydrate lacto - n - fucopentaose iii (lnfpiii), which induced high levels of il-4, il-5, and il-10 by nasal lymphocytes bypassing the need for an adjuvant. dcs are also targeted by lyso - phosphatidylserine (lyso - ps) present in sea, to dampen the th2 response. lyso - ps was shown to act on dcs via the tlr2 pathway, endowing them with the ability to induce il-10-producing treg cells. whole sea, as well as sea - derived molecules, has been described to interfere with animal models of allergy - related diseases. yang et al. used s. japonicum sea to test its effect in experimentally induced asthma in mice. sea increased il-10 production and expression of foxp3 on cd4cd25 t cells, both with immunosuppressive activity, while at the same time decreasing the expression of th2 (il-4 and il-5) cytokines, suppressed antigen - induced airway inflammation, recruitment of inflammatory cells into the lungs, and development of asthma. s. mansoni egg - secreted chemokine - binding protein (smckbp) was reported to significantly diminish ear swelling of hapten - sensitized mice in a contact hypersensitivity model. isolation of the inflammatory infiltrate from hapten - treated ears of smckbp - treated mice showed reduction of neutrophilia when compared to cells isolated from the ears of control animals. showed that smckbp has specific in vivo activity, suppressing immediate or local inflammation. additionally, three s. mansoni antigens sm22.6 (soluble protein associated with the s. mansoni tegument), piii (a multivalent antigen of s. mansoni adult worms), and sm29 (a membrane bound glycoprotein on the s. mansoni adult worm tegument) were evaluated in the ova - induced airway inflammation mouse model. all three antigens showed a beneficial immunomodulatory effect in this allergy model characterised by suppression of airway inflammation, reduced eosinophilia in the lungs, decreased ova - specific ige levels, and lower th2-specific cytokine production in bal when compared to asthmatic animals. sm22.6 significantly induced higher levels of il-10, while both sm22.6 and piii lead to increased expression of cd4foxp3 t cells suggesting that treg cells might be involved in the modulation of this aberrant th2 inflammation. although ascaris infections are described to support allergic inflammation (section 2.3), products from a. suum have also been described to contain immunomodulatory activity, in particular the 200 kda protein pas-1. administration of pas-1 to mice that had been injected with lps into sterile air pouches ed in a significant decrease in neutrophil migration as well as suppressing proinflammatory cytokines il-6, tnf-, and il-1. itami et al. used a murine model of asthma induced by another a. suum - molecule, apas-3 that promotes ige production , to test the suppressive effect of pas-1. in this experimental model of asthma induced by apas-3, pas-1-treated mice showed decreased eosinophil migration into the lungs, lower il-4, il-5, ccl11 and rantes production in the bal, as well as reduced airway hyperreactivity. moreover, il-10 production was considerably increased after pas-1 treatment when compared to apas-3-immunized controls. thus, a. suum has allergenic components as well as suppressive products that downregulate allergic responses. more recently, a study from the same group showed suppressive effect of pas-1 also in ova - induced airway inflammation. in line with previous findings, pas-1 reduced eosinophilia, decreased production of th2 associated cytokines in the bal and ova - specific ige and igg1 levels in serum. moreover, the effect was observed in il-12- but not in il-10- and ifn--deficient animals, thus il-10 and ifn- played a role in the immunomodulatory effect of pas-1. furthermore, the authors reported that the immunomodulatory effect of pas-1 was mediated by cd4cd25 and cd8 t cells as shown by adoptive cell - transfer into ova - sensitized and challenged mice. recipients of pas-1-primed cd4cd25 t cells had increased levels of il-10/tgf- whereas mice with transferred pas-1-primed cd8 t cells showed prominent ifn- production. those three cytokines were described to play important roles in the suppression of allergic diseases and reported to be strongly induced during successful allergen - specific immunotherapy in humans as reviewed by holgate and polosa. helminths have had millions of years to evolve regulatory mechanisms that circumvent the mammalian immune response and allow for the longevity characteristically observed during infection. thus, understanding of this coevolutionary nature is important in order to comprehend the immunomodulatory potential of helminth - derived molecules. this will better allow the development of therapeutical targets in symptomatic disease in helminth infections and help to break transmission of infection in endemic countries. furthermore, the genetic basis underlying differences in responses of different individuals to helminth infections should be examined to develop a full understanding of these parasites, and current studies are beginning to address this. in particular, relevant experimental models for filarial disease are required to study both asymptomatic and pathological responses. the epidemiological data reported in this paper together with experimental studies clearly support the view of the immunomodulatory potential of helminth - derived products to interfere with and even prevent aberrant allergic inflammation. research should focus on studying the immunomodulatory effects of helminth - derived products on allergy and strive to develop new therapeutic strategies by identifying the mechanisms and pathways utilised by such molecules in mediating their immunomodulatory effects.

helminths are master regulators of host immune responses utilising complex mechanisms to dampen host protective th2-type responses and favour long - term persistence. such evasion mechanisms ensure mutual survival of both the parasite and the host. in this paper , we present recent findings on the cells that are targeted by helminths and the molecules and mechanisms that are induced during infection. we discuss the impact of these factors on the host response as well as their effect in preventing the development of aberrant allergic inflammation. we also examine recent findings on helminth - derived molecules that can be used as tools to pinpoint the underlying mechanisms of immune regulation or to determine new anti - inflammatory therapeutics.

while opportunistic infections continue to be enameled care of patients infected with acquired immunodeficiency, cancer remains overlooked in the care of these patients in developing countries. according to center disease control (cdc), the revised definition of aids criteria includes the biological criteria (cd4 below 200) and/or clinical criteria (opportunistic infections, cancers of the cervix, kaposi sarcoma, and non - hodgkin lymphoma) associated with infection by human immunodeficiency virus (hiv). the occurrence of malignant tumors in patients with hiv infection currently represents for years to come one of the most difficult and controversial challenges of therapeutic management of these patients. as of 2011 africa was the epicenter of viral related cancers even prior to the hiv epidemic (e.g., kaposi 's sarcoma ( ks), burkitt's lymphoma, cervical cancer, and hepatocellular carcinoma ). indeed, 30 to 40% of patients infected with hiv are likely to develop cancer about their disease; the incidence of certain cancers such as kaposi's sarcoma has decreased since the introduction of potent antiretroviral therapies; other malignancies associated with aids do not seem to be affected by the establishment of this new therapeutic strategy. the prevalence coinfection tb / hiv (25.5%) is well known while no study on cancers associated with hiv has been performed in our country. the widespread use of antiretroviral therapy (arv) in industrialized countries since the mid-1990s led to a dramatic decrease in mortality of patients with hiv infection and incidence of opportunistic infections. however, antiretroviral treatment types haart also had a significant impact on the natural history of hiv - associated malignancies. a us study found an increase in median survival of aids patients from 11.3 months before 1985 to 12.5 months in 1986 to 20.8 months in 1987 for aids cases diagnosed in washington. according to a recent british study, 40% of aids patients have cancer during the course of their disease. in france, a study showed that, out of a total of 964 recorded deaths, 149 were attributed to the type of aids defining cancers. according to the regional office for africa of who sub - saharan african countries have experienced nearly 600,000 deaths in 2008 related to tumors. if this epidemiological trend continues due to longer life expectancy and increased exposure to tobacco, africa will have by 2020 from 800,000 to 1,000,000 new cases of cancer each year. the cancer incidence rate is estimated at 95.5 cases per 10 inhabitants in west africa and 90.0 per 10 in guinea. as in most sub - saharan african countries, there is very little data available on the prevalence of hiv infection in cancer patients in west africa, especially in our country. this study aims to determine the prevalence of hiv infection among patients seen at the surgical oncology unit of donka. data from this study were obtained from the hospital and came from surgical oncology unit of donka national hospital, teaching hospital of conakry (guinea). the study involved the records of cancer patients followed with hiv infection from may 2007 to december 2012. this checkup has become routine for any cancer patient received in our unit since january 2009. the prevalence has been determined according to the number of patients and pathology subject of consultation or follow - up. pathologies associated with hiv have been classified into malignant, benign, and inflammatory diseases. social characteristics (age, gender, marital status, tobacco and alcohol consumption, education, and body mass index) and immune status (hiv type, cd4 count, and antiretroviral treatment) were reviewed according malignant tumor pathologies, divided into defining aids and nondefining aids cancers. gynecological history (age at menarche, first full pregnancy) was determined in women with breast cancer. out of 3143 patients received from may 2007 to december 2012, 69 (2.2%) tested positive for hiv in our unit. this proportion was 12 (1.9%) of 625 patients from 2007 to 2008 and 57 (2.3%) of 2518 patients from 2009 to 2012. of the 64 hiv - infected patients, 54 (78.3%) had a malignant tumor, 8 (11.6%) of benign and 7 (10.1%) of inflammatory tumors (table 1). the prevalence of hiv in cancer patients was 54 on 2598 cases (2.1%). of the 54 cases of cancers associated with hiv, there were 11 (20.4%) defining aids and 43 (79.6%) nondefining aids. cancer patients had a median age of 39 (iqr 3148) against 32 (iqr 2538) for noncancer patients. this median age was different about defining aids status: 33 (25.040.0) versus 39 (iqr 35.750.0) (p = 0.007). their body mass index was 20,4 kg / m (iqr 17.721.8). they were unschooled in 40 (74.1%) and married in 35 (64.8%). tobacco use was found in 13 (24.1%) and alcohol consumption in 14 (20.2%). cd4 count, performed in 22 patients, showed a median of 317 cells / ml (iqr 246.2534.2). antiretroviral treatment was performed in 40 (74.1%) patients of which it was performed in 39 only after the cancer diagnosis. table 2 shows the distribution of different types of cancers depending on the aids definition. among the cancers defining aids, non - hodgkin's lymphoma (11.1%) and cervical cancer (9.3%) were the most common. breast cancer (11.1%), followed by cancer of the liver (11.1%), and eye and annexes (11.1%) were leading nondefining aids cancers. patients with breast cancer had a median age of 14.0 years (iqr 12.515.0) at menarche and 22.0 years (iqr 17.025.0) in the first full term pregnancy; and they had 1.0 full pregnancy in term (iqr 0.53.0). this preliminary study shows a high prevalence (2.1%) of the hiv infection in patients with cancer in our surgical oncology unit. this prevalence appeared to be higher than in the general population in guinea (1.5%) but lower than in the tertiary hospital (6.0%) in nigeria. risk of cancer occurrence in hiv - infected people is 6.39 times higher compared to uninfected hiv people. in this study, non - aids defining cancers were more frequent than defining aids cancers (77.8% versus 22.2%). this could be explained partly by the decrease in the number of cancer cases defining aids since the beginning of antiretroviral therapy as supported by yanik et al. in the us. also, kaposi's sarcoma (ks) and cervical cancer are mainly managed, respectively, by the dermatology and gynecology departments of teaching hospital. kaposi's sarcoma is very common in dermatology and the guinean first data were published by cisse et al.. in nigeria, ks and cervical cancer are the most common cancers among non - aids defining cancers. cervical cancer is the most common cancer in guinea with a high prevalence of human papillomavirus infection. in this study, nhl was leading defining aids cancer. this predominance of breast cancer among nondefining aids cancers is reported by studies in sub - saharan africa. the hiv infection is very prevalent, about 35.4% among women with breast cancer in uganda and 19.7% in south africa. in developed countries, lung cancer is the most common of nondefining aids cancers. conversely, those with aids classifying cancer were younger than those who had a non - aids defining illness cancer. the female gender and the young age of patients are already reported by several authors. this could be related to the irregular availability of reagents for cd4 and antiretroviral despite their free access in our country. cd4 count, carried out for 22 cases out of 54, showed no difference in the depth of immunosuppression that cancer is either aids defining or not. for defining aids cancers , there is no doubt that the risk is higher in the case of lower cd4 count (< 200 cells / ml). however, there are controversies with regard to the depth of immunosuppression in nondefining aids cancer. we consider that the art coverage rate (74.1%) is low in this category of the population infected with hiv, although this rate is higher than the rate of coverage in guinea (50.0%). these patients are faced with two problems: hiv / aids with the supported opportunistic infections and cancer that is not supported by the national program against aids. that is why a national prospective study is needed to determine the true prevalence of cancer among people living with hiv. it will also be important to determine whether hiv changes the risk factors, clinicopathological futures, and prognosis of these cancers. breast cancer is the most common in this association. a national survey of a large sample is needed to determine the true prevalence and impact of hiv on cancer prognosis.

aim. to determine the prevalence of hiv infection among patients seen at the surgical oncology unit of donka (conakry, guinea). method. we conducted a retrospective and descriptive study of hiv infection in cancer patients from may 2007 to december 2012. social characteristics (age, gender, marital status, and education) and immune status (hiv type, cd4 count) were reviewed. . out of 2598 cancer patients, 54 (2.1%) tested positive for hiv. there were 11 (20.4%) defining aids and 43 (79.6%) nondefining aids cancers. the most frequent cancers were breast (26.0%), non - hodgkin lymphoma (11.1%), liver (11.1%), eye and annexes (11.1%), and cervical cancer (9.3%). these patients were female in 34 (63.0%) and had a median age of 39 years and body mass index was 20,3 kg / m2. they were unschooled in 40 (74.1%) and married in 35 (64.8%). cd4 count showed a median of 317 cells / ml. antiretroviral treatment was performed in 40 (74.1%). . hiv prevalence is higher in patients in our unit of surgical oncology. breast cancer was the most common in this association. a national survey of a large sample is needed to determine the true prevalence and impact of hiv on cancer prognosis.

interest in tomosynthesis and its clinical applications has been revived by recent advances in digital x - ray detector technology. in tomosynthesis, an x - ray tube and an x - ray receptor tomosynthesis captures only one set of discrete x - ray projections that can be used to reconstruct any plane of the object retrospectively. this article will review and identify indications for x - ray digital linear tomosynthesis (dlt). the chest phantom (n-1 type, kyoto kagaku co., tokyo, japan) and the artificial pulmonary nodules of type n1 are depicted in the images. the artificial pulmonary nodules were of the ground - glass opacity type (5 and 7 mm), and the spheres were of the same diameter and composition. for the dual - energy evaluation study, calcification was simulated using hydroxyapatite (powder type). the relationship between the radiation attenuation properties of glass and those of real pulmonary nodules was correlated. tomosynthesis (sonialvision safire ii, shimadzu co., kyoto, japan) and ct (64-slice somatom definition scanner ; siemens medical systems, forchheim, germany) images of the artificial pulmonary nodules (ground - glass opacity type). the quality of the images obtained by tomosynthesis imaging were clearer than those obtained by ct (ct slice thickness : 5 mm). des - tomosynthesis and des radiography (sonialvision safire ii) of the same slice, demonstrates the content of the artificial pulmonary nodules with and without calcification. the high - contrast detectability phantom case having a clear, contrast detectability on des - tomosynthesis imaging produced for identical planes (tomosynthesis acquisition angle : 40). owing to its high sensitivity, normal - dose helical computed tomography (ct) is currently considered the gold standard for lung cancer detection. early reports indicate that low - dose helical ct has the potential to detect early lung cancer, and thus decrease morbidity. ct has the advantage of improved detection of lesion by eliminating visual clutter of overlying anatomy. however, there are disadvantages of using ct when compared with chest radiography, such as high - radiation dose and cost. on the other hand, although chest radiography has advantages such as short examination time, low cost, and easy access, there are also important disadvantages such as low sensitivity and specificity. in chest radiography, the three - dimensional chest is projected onto a two - dimensional image. consequently, the ability to detect pathological findings is limited by the overlapping anatomy rather than by quantum noise. chest radiography has been shown to have a relatively low sensitivity for the detection of pulmonary nodules. the poor sensitivity of chest radiography precludes its use as a screening modality, despite its advantages of low cost, low dose, and wide distribution of devices. dlt is a method that provides some of the tomographical benefits of ct at a reduced radiation dose and cost and with an approach that can be easily implemented in conjunction with chest radiography. current state of dlt originates from the older technique of geometric tomography, which has largely fallen out of favor in chest imaging owing to positioning difficulty, high radiation dose, and residual blur from out - of - plane structures. dlt overcomes the difficulties of geometric tomography by permitting reconstruction of numerous slices of the image from a single low - dose acquisition of image data. although improving detection of pulmonary nodules may be an early area of emphasis for the application of tomosynthesis, it also has potential for use in other areas of thorax imaging. presence of calcification and stability of the mass over 2 years suggest its benign nature. a benign pattern of the calcifications has been considered necessary to exclude malignancy. in the evaluation of diffusely disseminated pulmonary nodules, identification of diffusely disseminated pulmonary nodules and calcifications in the nodules has been helpful in limiting the differential diagnosis. conventional radiography and conventional tomography have been used to detect calcifications, but they have been largely replaced by ct. however, ct has several inherent problems, including motion artifacts and the variety of reconstruction algorithms that are used by different ct scanners. dual energy subtraction (des) imaging has been proposed and investigated by many researchers as a means of reducing the impact of anatomic noise on disease detection by chest radiography. des involves making two radiographical projections of the patient using different energy x - ray beams. by exploiting the difference in the energy dependence of attenuation between bone and soft tissue, the bone contrast can be reduced to produce a soft tissue image and the contrast of the soft tissue can be reduced to produce a bone image. des digital radiography has been found to be useful in detecting calcifications within pulmonary masses. main drawback of the des technique, is that its projections may in overlap of anatomical features (e.g., calcifications superimposed over the ribs or spine). des dlt is a new technique, and therefore there is no guidance for its integration into the clinical practice of chest radiography. the most reliable signs for discriminating between benign and malignant masses are the growth rate of the mass and presence or absence of calcifications within the mass. since calcifications are commonly observed in benign masses and no other radiographical characteristic is specific in characterizing a mass, it is important to detect and characterize calcification within lesions. using des dlt, the presence, distribution, and characteristics of calcifications in lung nodules can be assessed to an extent that is not possible with currently available ct imaging and projection - type des techniques. in addition, this technique is not susceptible to the problems of image overlap, partial volume effect, or shifting of the image plane. calcified tuberculous foci in a 74-year - old man. comparison of the chest images obtained with x - ray radiography, digital tomosynthesis, des - tomosynthesis, and high - resolution ct (hr - ct). des - tomosynthesis and hr - ct appear to have similar accuracy in detecting calcification in the pulmonary nodules. ideally, structures in a given plane of interest should be clearly displayed in the corresponding tomosynthesis reconstruction plane, whereas structures located outside of that plane should not be visible. essentially, the limited angular range of the tomosynthesis image acquisition geometry dictates that spatial resolution is limited in the dimension perpendicular to the detector plane. as a , out - of - plane structures can not be completely removed from the reconstruction plane and are present in every such plane. however, most of these structures are not visible because various low - amplitude structures from projections overlap in the reconstruction plane, and therefore, appear blurred. out - of - plane structures from high - attenuation features can not be blurred and appear as multiple replicates in every reconstruction plane, except for the one in which the actual high - attenuation feature (e.g., rib) is located. at one projection angle, these ghosting features are distributed along the line made by the x - ray source and the actual feature. out - of - plane structures are present in every reconstruction plane, but most are not visible because the various low - amplitude structures from projections overlap each other in the reconstruction plane, and therefore are blurred. tomosynthesis is worthy of further evaluation because it is flexible and can accurately reproduce the shape of an artificial pulmonary nodule. tomosynthesis can be considered the modality of choice to detect and investigate simulated artificial pulmonary nodules with and without calcification.

the purpose of this paper is to identify indications for volumetric x - ray digital linear tomosynthesis (dlt) with single- and dual - energy subtraction techniques for artificial pulmonary nodule detection and compare x - ray dlt, x - ray digital radiography, and computed tomography.

endothelial dysfunction causes different disorders in human body including preeclampsia during pregnancy. the pathogenesis may be due to inflammatory responses that trigger pregnancy - induced hypertension consequently preeclampsia complicates up to 510 and 3% of pregnancies in developed and developing countries, respectively. it plays an important role in occurring prenatal and perinatal events including premature delivery, severe hypertension, increasing the risk of stroke, maternal death. mothers with the medical history of preeclampsia are predisposed to for cardiovascular diseases due to endothelial dysfunction may be persistent after delivery. several studies reported the role of endothelial cells in endothelial - dependent diseases including preeclampsia and it is recently proved that endothelial dysfunction plays an important role in pathogenesis of preeclampsia. the endothelial function should be evaluated by measuring brachial artery flow - mediated dilation (fmd) by high - resolution b - mode ultrasonography after triggering shear stress. reported that low - dose aspirin, as an antiplatelet, in early pregnancy decrease the risk of preeclampsia and intrauterine growth retardation (iugr). another study represented that low - dose aspirin decreases the risk of preeclampsia in healthy nulipar pregnant women but has no impact on prenatal morbidity. although there are lots of surveys on preeclampsia, a few studies evaluate the effect of asa in preeclamptic patients. the aim of this study is to evaluate the effect of low - dose acetylsalicylic acid (asa) on endothelial dysfunction in preeclamptic patients. this trial has been registered at http://www.irct.ir/ registration with i d number irct2016040527135n2 and has been approved by the isfahan university of medical sciences ethical board. in this randomized clinical trial, pregnant women with documented preeclampsia on the basis of national high blood pressure education program working group on high blood pressure in pregnancy, 2000, who visited in st - zahra hospital, isfahan, iran, patients with any clinical evidences of diabetes mellitus, pernicious anemia, aplastic anemia, megaloblastic anemia, known cardiovascular disease, hypercholesterolemia, hypertriglyceridemia, renal failure, thyroid disease, history of pregnancy - induced hypertension, malignancy, liver dysfunction, infectious disease, migraine, and stroke did not include in this study. in addition, patients with the history of twin pregnancy, smoking cigarette, alcohol consumption, and drug abuse did not include in the study. women who sensitize to the drug (vertigo, insomnia, rash, severe cough, angioedema, purities and bronchospasm, and drug consumption), poor compliance to use prescribed drug and hygiene advice and who use drugs < 2 months during pregnancy excluded from the study. enrolled patients were divided randomly (allocation ratio equal to 1:1) into two groups by random allocation service (www.saghaei.net/ra/). asa80 mg (osveh, tehran, iran) or placebo was taken daily by oral administration from the initiation of diagnosis until 2 months after delivery by the trial participant. every patient's fmd were evaluated at the beginning and 2 months after delivery with the same experienced operator at a same period of the time (35 pm) by high - resolution b - mode ultrasonographic (sonoace, deutschland). fmd estimated by the following formula: fmd (%) = (/bbd) 100 basal brachial diameter (bbd) is measured in resting position, and postocclusion diameter (pod) is measured 30 s before and 90 s after cuff evacuation (cuff pressure is increased up to 300 mmhg for 5 min before evacuation). potential confounding variables included in the independent samples t - test were maternal age, parity, prepregnancy body mass index (bmi), and abortion. t - test or mann whitney test will be used in the comparison of means of birth weight and gestational age, between the intervention and placebo groups. to compare fmd in each group, before and after the intervention, paired t - test was used by spss software, version 21, chicago, il, usa (p < 0.05 was decided significant). this trial has been registered at http://www.irct.ir/ registration with i d number irct2016040527135n2 and has been approved by the isfahan university of medical sciences ethical board. in this randomized clinical trial, pregnant women with documented preeclampsia on the basis of national high blood pressure education program working group on high blood pressure in pregnancy, 2000, who visited in st - zahra hospital, isfahan, iran, patients with any clinical evidences of diabetes mellitus, pernicious anemia, aplastic anemia, megaloblastic anemia, known cardiovascular disease, hypercholesterolemia, hypertriglyceridemia, renal failure, thyroid disease, history of pregnancy - induced hypertension, malignancy, liver dysfunction, infectious disease, migraine, and stroke did not include in this study. in addition, patients with the history of twin pregnancy, smoking cigarette, alcohol consumption, and drug abuse did not include in the study. women who sensitize to the drug (vertigo, insomnia, rash, severe cough, angioedema, purities and bronchospasm, and drug consumption), poor compliance to use prescribed drug and hygiene advice and who use drugs < 2 months during pregnancy excluded from the study. enrolled patients were divided randomly (allocation ratio equal to 1:1) into two groups by random allocation service (www.saghaei.net/ra/). asa80 mg (osveh, tehran, iran) or placebo was taken daily by oral administration from the initiation of diagnosis until 2 months after delivery by the trial participant. every patient's fmd were evaluated at the beginning and 2 months after delivery with the same experienced operator at a same period of the time (35 pm) by high - resolution b - mode ultrasonographic (sonoace, deutschland). fmd estimated by the following formula: fmd (%) = (/bbd) 100 basal brachial diameter (bbd) is measured in resting position, and postocclusion diameter (pod) is measured 30 s before and 90 s after cuff evacuation (cuff pressure is increased up to 300 mmhg for 5 min before evacuation). potential confounding variables included in the independent samples t - test were maternal age, parity, prepregnancy body mass index (bmi), and abortion. t - test or mann whitney test will be used in the comparison of means of birth weight and gestational age, between the intervention and placebo groups. to compare fmd in each group, before and after the intervention, paired t - test was used by spss software, version 21, chicago, il, usa (p < 0.05 was decided significant). eighty - two preeclamptic pregnant patients were enrolled in this study between february 2015 and february 2016. the mean of patients ages was 31.03 4.21 years (with a range of 2141 years). as shown in table 1, patients have no significant differences in age, bmi, level of serum triglycerides, low - density lipoprotein, high - density lipoprotein, fasting blood sugar, and hemoglobin. demographic and baseline characters of patients the mean value of fmd in intervention (9.61 5.58) and control group (9.40 4.33) have no significant differences before consumption of drugs (p = 0.089). as demonstrated in table 2, fmd increased significantly in intervention group after daily consumption of 80 mg of asa. in control group, in contrast, fmd has shown no significant differences before and after daily consumption of placebo. flow - mediated dilation in each group before and after consumption of drug / placebo meanwhile, fmd in intervention group significantly increases after daily consumption of 80 mg asa in comparison with control group after daily consumption of placebo. in this clinical trial on pregnant women suffering from preeclampsia visited in st - zahra hospital, isfahan, iran, 82 enrolled patients were divided randomly into two groups who received asa 80 mg or placebo daily by oral administration from initiation of diagnosis until 2 months after delivery. our showed that daily oral administration of low - dose asa increase fmd significantly which means it can improve endothelial function. several experimental studies approved the effect of asa on platelet function as a potent antioxidant. these surveys showed asa can affect endothelial function through oxidative stress pathway in vascular inflammation process. fmd is inversely correlated to platelet reactivity in both controls and acute myocardial infarction patients. this study showed that platelet adhesion and aggregation in inflammation state lead to endothelial dysfunction and platelet reactivation decrease endothelial dilation. in support of this study, raghavan et al. also reported that antiplatelet treatment can reduce vascular dysfunction. magen et al. in a study at 2005 showed that addition of low - dose aspirin to antihypertensive treatment and statins in hypertensive and hypercholesterolemic patient can reduce systolic and diastolic blood pressure through improved no - mediated endothelial function. similar effects of low - dose aspirin on blood pressure and reduce risk of preeclampsia were seen in a study on pregnant women in houston, and suggest that aspirin administration in pregnancy are useful for high - risk mothers and are safe for fetus. endothelial dysfunction due to inflammation process and imbalance between vasoconstrictor and vasodilator modulates are known pathophysiology of preeclampsia. in endothelial disorders, such as preeclampsia, poor placental perfusion leads to activation of platelets and the clotting cascade, ing in an imbalance among prostacyclin (vasoactive modulates in blood flow and inhibit aggregation) with thromboxane a2 (acting as a vasoconstrictor and promoting platelet aggregation). increased thromboxane and reduced prostacyclin levels are associated with infarction and thrombotic vasculopathy, which caused preeclampsia and its adverse features outcomes. schramm et al. in a study on aspirin effect for the prevention of preeclamsia in lupus pregnant patient reported that low - dose aspirin (6080 mg / day) may prevent preeclampsia by modulating the thromboxane a2/prostacyclin ratio to optimize placental blood flow and prevent placental thrombosis while aspirin will not eliminate all cases of preeclampsia, it is currently the best and safest available drug for influencing the pathogenesis and clinical presentation of preeclampsia. in this study, aspirin treatment had a risk reduction of up to 20% for preeclampsia development in lupus patient. furuno et al. in a survey about the effects of various doses of aspirin on platelet activity and endothelial function indicate that aspirin at any daily dose over 81 mg suppressed platelet activity, and the optimal dose of endothelial function was 162 mg / day. however, 660 mg / day of aspirin worsened endothelial function. this study explains aspirin plays an important role in the primary and secondary prevention of cardiovascular events and it has remained the most cost - effective clinical drug for over three decades. a high dose of aspirin achieves both platelet inhibition and vasodilation, whereas a low dose spares endothelial cyclooxygenase activity and vasodilatation. pregnant women reported prescription of aspirin (60150 mg / day) to this group, appears to be effective in reducing the prevalence of early pe. aspirin also can reduce the risk of preterm delivery, iugr, and prenatal death. study showed similar effects and suggested that low - dose aspirin initiated at 16 weeks of gestation is associated with a greater reduction of perinatal death and other adverse perinatal outcomes than when initiated at > 16 weeks. first and most important limitation of our study is small sample size due to difficulties to diagnosis pure preeclamptic patients who met inclusion criteria. the last but not least was an unequal period of drug administration by participants due to emergency indications for cesarean section which some of the participants did meet. first and most important limitation of our study is small sample size due to difficulties to diagnosis pure preeclamptic patients who met inclusion criteria. the last but not least was an unequal period of drug administration by participants due to emergency indications for cesarean section which some of the participants did meet. this study demonstrated an association between low - dose aspirin and increase flow - mediated vasodilation in the brachial artery in preeclamptic patient. increase mean of fmd in groups who took regular daily oral 80 mg aspirin starting right at the initiation of diagnosis until 2 months after delivery shows that this drug through biological pathway can improve endothelial function and can be significantly affected maternal blood pressure during pregnancy and some endothelium - dependent disease such as preeclampsia and its associated adverse outcomes. this study was supported by isfahan university of medical sciences, isfahan, iran ( grant no. this study was supported by isfahan university of medical sciences, isfahan, iran ( grant no. fb contributed in the conception of the work, conducting the study, revising the draft, approval of the final version of the manuscript, and agreed for all aspects of the work. mh contributed in the conception of the work, drafting and revising the draft, approval of the final version of the manuscript, and agreed for all aspects of the work. ez contributed in the conception of the work, drafting and revising the draft, approval of the final version of the manuscript, and agreed for all aspects of the work. kh contributed in the conception of the work, drafting and revising the draft, approval of the final version of the manuscript, and agreed for all aspects of the work. sb contributed in the conception of the work, drafting and revising the draft, approval of the final version of the manuscript, and agreed for all aspects of the work.

: preeclampsia complicates up to 3% of pregnancies in developing countries. endothelial dysfunction plays an important role in pathogenesis of preeclampsia. in this study , we aim to evaluate the effect of low - dose aspirin on endothelial dysfunction in preeclamptic patients.materials and methods: in this triple - blind randomized clinical trial, enrolled patients were divided randomly into two groups. acetylsalicylic acid (asa) 80 mg or placebo will be taken daily by oral administration from the initiation of diagnosis until 2 months after delivery. every patient's flow - mediated dilation (fmd) were evaluated at the beginning of study and 2 months after delivery with the same experienced operator at a same period of the time (35 pm) by high - resolution b - mode ultrasonographic. t - test or mann whitney test was used in the comparison of means between the intervention and placebo groups. to compare fmd in each group, before and after the intervention, paired t - test was used.:mean value of fmd in intervention (9.61 5.58) and control group (9.40 4.33) have no significant differences before drug consumption (p = 0.089). fmd in intervention group significantly increased after asa consumption .: increase mean of fmd in intervention group shows that this supplement can improve endothelial function.

eyfp - actin and eyfp - nls - actin expression plasmids were kindly provided by primal de lanerolle (university of illinois, chicago). eyfp tagged actin behaves like an endogenous actin and has been used to visualize actin dynamics in the inner cells. we have confirmed that the expressed eyfp - nls - actin accumulates in the nucleus and forms nuclear f - actin bundles. hela cells were cultured in dulbecco's modified eagle's medium (gibco) supplemented with 10% fetal bovine serum and antibiotics (penicillin and streptomycin) at 37 c in a 5% co2 humidified atmosphere. transfection of hela cells was performed using lipofectamine 2000 (invitrogen) and 2 g of plasmid dna. captured images of hela cells expressing the eyfp - actin or the eyfp - nls - actin are shown in fig. 1. total rna was isolated from the eyfp - actin or eyfp - nls - actin expressing hela cells by using an rneasy mini kit (qiagen) according to the manufacturer's protocol. the quality of isolated rna was assessed by monitoring the rna integrity number (rin). we confirmed that the rin scores of the sample rnas isolated from the eyfp - actin or eyfp - nls - actin expressing hela cell were 10. rna labeling was performed using a low input quick amp labeling kit (one - color). 0.6 g of cyanin-3 (cy3)-labeled crna probe, prepared from 100 ng of total rna, was fragmented and hybridized to the agilent-039494 sureprint g3 human ge v2 8x60k microarray (gpl16699) using a gene expression hybridization kit (agilent) according to the manufacturer's instructions. after hybridization, the array was scanned using an agilent dna microarray scanner (g2565ca). the agilent feature extraction software (agilent) was used to extract raw data from the scanned array images. these raw data files were registered as geo (gse59799). a scatter plot, showing normalized signal intensity values of different genes in eyfp - actin and eyfp - nls - actin expressing hela cells, is shown in fig. as shown, the scatter plot exhibited good correlation coefficient (r = 0.9859) between these signal intensities. an ma plot is used to identify systematic variations within the arrays, where m is the log ratio (log2 ( exp . gscale signal) log2 (base gscale signal) ) and a is the average log of the product of the two intensities [{ log2 ( exp . as shown, the number of up - regulated genes was greater than the number of down - regulated genes, suggesting that the nuclear actin is mainly involved in gene activation rather than in gene repression . eyfp - actin and eyfp - nls - actin expression plasmids were kindly provided by primal de lanerolle ( university of illinois, chicago). eyfp tagged actin behaves like an endogenous actin and has been used to visualize actin dynamics in the inner cells. we have confirmed that the expressed eyfp - nls - actin accumulates in the nucleus and forms nuclear f - actin bundles. hela cells were cultured in dulbecco's modified eagle's medium (gibco) supplemented with 10% fetal bovine serum and antibiotics (penicillin and streptomycin) at 37 c in a 5% co2 humidified atmosphere. transfection of hela cells was performed using lipofectamine 2000 (invitrogen) and 2 g of plasmid dna. captured images of hela cells expressing the eyfp - actin or the eyfp - nls - actin are shown in fig. 1. total rna was isolated from the eyfp - actin or eyfp - nls - actin expressing hela cells by using an rneasy mini kit (qiagen) according to the manufacturer's protocol. the quality of isolated rna was assessed by monitoring the rna integrity number (rin). we confirmed that the rin scores of the sample rnas isolated from the eyfp - actin or eyfp - nls - actin expressing hela cell were 10. rna labeling was performed using a low input quick amp labeling kit (one - color). 0.6 g of cyanin-3 (cy3)-labeled crna probe, prepared from 100 ng of total rna, was fragmented and hybridized to the agilent-039494 sureprint g3 human ge v2 8x60k microarray (gpl16699) using a gene expression hybridization kit (agilent) according to the manufacturer's instructions. after hybridization, the array was scanned using an agilent dna microarray scanner (g2565ca). the agilent feature extraction software (agilent) was used to extract raw data from the scanned array images. these raw data files were registered as geo (gse59799). a scatter plot, showing normalized signal intensity values of different genes in eyfp - actin and eyfp - nls - actin expressing hela cells, is shown in fig. as shown, the scatter plot exhibited good correlation coefficient (r = 0.9859) between these signal intensities. an ma plot is used to identify systematic variations within the arrays, where m is the log ratio (log2 ( exp . gscale signal) log2 (base gscale signal) ) and a is the average log of the product of the two intensities [ { log2 ( exp. 2b. as shown, the number of up - regulated genes was greater than the number of down - regulated genes, suggesting that the nuclear actin is mainly involved in gene activation rather than in gene repression. herein, we describe the nuclear actin - mediated regulation of gene expression in hela cells by using a commercially available rna microarray. our microarray analysis data will be useful for elucidating the role of nuclear actin in transcription regulation.

actin, an integral component of the cytoskeleton, plays crucial roles in a variety of cell functions, including cell migration, adhesion, polarity and shape change. studies performed during the last couple of decades have revealed that the actin also exists in the nucleus. however, the function and properties of nuclear actin remained elusive so far. recently, we showed that an actin tagged with eyfp and fused with a nuclear localization signal (eyfp - nls - actin) formed visible filamentous (f)-actin bundles in cells. to obtain further details about the individual genes that are affected by the nuclear actin , we have used the microarray analysis to determine the changes in the expression levels of rnas in hela cells as a of eyfp - nls - actin expression. our suggest that the nuclear actin plays a role in the activation of genes rather than their repression. the data has been deposited in the gene expression omnibus (geo) database under the accession number gse59799.

urticaria is a condition characterized by localized or widespread pruritic wheals that typically exist for no more than 24 h. by definition acute urticaria lasts no longer than six weeks, whereas chronic urticaria lasts longer, often several years. autoimmune urticaria is not a well - defined term but it is generally recognized that those with autoimmune urticaria have anti - igg antibodies against the high - affinity ige receptor (fceri) on mast cells and basophils or directly to ige antibodies. h1 antihistamines are recommended as first - line therapy for chronic urticaria; leukotriene receptor antagonists are indicated as second - line therapy, whereas immunosuppressive drugs such as corticosteroids, azathioprine or cyclosporine a should be reserved for severe recalcitrant disease. omalizumab is a recombinant humanized monoclonal antibody that blocks the high - affinity fc receptor of ige.. however, there is currently more and more data showing promising in the management of patients suffering from other allergic conditions such as chronic urticaria. we present a case series of chronic urticaria patients in a university department treated with omalizumab and give an overview of the existing literature concerning omalizumab treatment of therapy - resistant chronic urticaria. the cases reported herein were selected consecutively from the department of dermatology at bispebjerg hospital in copenhagen. all patients were initially referred to the department with a diagnosis of urticaria and were considered eligible for this report if they began treatment with omalizumab for urticaria during the one - year period from november 2010 to october 2011. for each case, the type and duration of urticaria was recorded as well as any previous medical treatment. if available, the of relevant serological markers including serum total ige and the urticaria hr test were noted. a histamine release > 16.5% was regarded as positive (reflab, copenhagen, denmark). all patients were treated with omalizumab at an initial dose of 150 mg once every two weeks, which was the department's standard dosing regimen. the clinical response to treatment with omalizumab was recorded and for each patient it was possible to score the individual response to treatment as: no response, partial response, or almost complete / complete resolution of symptoms during treatment. furthermore, the duration and any side effects of omalizumab were recorded. the response to treatment in our case series was compared with reports from the existing english language literature retrieved from pubmed using the search terms: omalizumab and anti - ige. cross - references were retrieved but this did not identify additional studies. a total of 19 patients (14 females) began treatment with omalizumab during the observation period (table 1). the mean age at the time of omalizumab initiation was 36 years for females and 49 for males. the mean duration of disease at initiation of omalizumab in the sample was 21 months for females and 24 months for males (one male patient had a duration of nine years). a total of 12 patients (63%) were classified as having chronic idiopathic urticaria, six patients (32%) had chronic autoimmune urticaria demonstrated by a positive urticaria hr test, whereas one patient had delayed pressure urticaria. all patients had antihistamine - resistant disease and all but two had been treated with other systemic drugs; 14 (74%) with prednisolone, 7 (37%) with azathioprine, cyclosporine a and/or mycophenolate mofetil, and 6 (32%) with tnf - a inhibitors. in total, 11 patients (58%) experienced almost complete or complete resolution of symptoms during treatment with omalizumab, whereas five (26%) experienced partial resolution; three patients (16%) had no benefit of the treatment and of these, one had to discontinue treatment due to side effects (nausea, headache and flu - like symptoms). however, in general, treatment was tolerated very well and only three patients (16%) reported side effects. these three patients were all females and among the youngest in the sample (15, 19 and 29 years of age). the present case series included 19 patients with therapy - resistant chronic urticaria. on an initial dose of omalizumab of 150 mg once every two weeks, a total of 84% of the patients experienced resolution of symptoms to a degree that exceeded the effect of previous treatments. the symptomatic effect of the treatment occurred in many of the patients after one or just a few days and no serious side effects were reported. these data add to the growing body of evidence supporting the use of omalizumab as a safe and effective treatment option for chronic urticaria. particularly, by december 2011, to our knowledge, a total of 59 cases of chronic urticaria treated with omalizumab have been reported in the literature comprising five cases of solar urticaria, two cases of heat urticaria, two cases of cold urticaria, three cases of delayed pressure urticaria, three cases of urticaria factitia, three cases of cholinergic urticaria, 40 cases of chronic idiopathic urticaria, and 16 cases of chronic autoimmune urticaria. furthermore, a total of 139 patients have been enrolled in two randomized controlled trials comparing omalizumab with placebo (table 2). the first randomized trial was restricted to patients with ige against thyroperoxidase; 27 patients were randomized to omalizumab and 22 to placebo. the absolute mean decrease in the urticaria activity score (uas) during the 24 weeks of treatment was 17.8 points in the experimental group and 7.9 points in the placebo group. according to the investigator's global assessment, as many as 67% of the patients in the experimental group were assessed as having achieved complete resolution of symptoms during the study compared with only 4% in the placebo group. the second trial included 90 patients with chronic idiopathic urticaria randomized to one of three different doses of omalizumab (75, 300 or 600 mg) or to placebo. the absolute mean decrease in the uas four weeks after a single dose of omalizumab was 14.6 points in the group receiving 600 mg omalizumab, 19.9 points in the group receiving 300 mg omalizumab, 9.8 points in the group receiving 75 mg omalizumab, and 6.9 points in the group receiving placebo. the mean decrease in the groups receiving 300 and 600 mg omalizumab was statistically significantly greater than in the placebo group. a total of 28.6, 36.0, and 4.4% of the patients had complete resolution of urticaria symptoms after receiving a single dose of 600, 300 and 75 mg omalizumab, respectively. both randomized trials reported mild but rather frequent side effects such as diarrhoea, headache, dysmenorrhoea and upper respiratory tract infections that, however, did not differ in prevalence between the groups receiving active treatment and placebo. the rapid improvement of urticaria symptoms reported in almost all previous cases treated with omalizumab indicates that ige plays an important role in chronic urticaria. however, although anti - ige mechanisms are thought to be the principal mode of action for omalizumab, several of the published cases support a role of other mechanisms such as induction of eosinophil apoptosis, downregulation of the inflammatory cytokines il-2, il-4, il-13 and tnf - a, increase in the activity of cd4 + cells by atp release, decrease in basophil releasability as well as a marked decrease in the expression of fceri. these observations offer an explanation for the successful also among patients with low levels of ige. the dosing of omalizumab for chronic urticaria is in many cases based on the recommendations for asthma, i.e. based on serum total ige levels and the weight of the patient. we saw a marked effect in most of our patients of treatment with 150 mg once every two weeks. in comparison, most of the previous cases were treated with a higher dose, predominantly with an initial dose of 300 mg, which was also the dose that produced the most marked clinical response in the experimental trials. on the contrary, earlier published case reports, in general, overestimated the effect of omalizumab compared with the controlled trials, and also did not report side effects or scored them as negligible or absent. despite an undeniable value of omalizumab for chronic urticaria, it is still not clear why the drug is effective in some patients and less so in others. nevertheless, the collective evidence points to omalizumab as a safe and effective treatment option for patients with chronic urticaria who do not sufficiently respond to standard therapy as recommended by existing guidelines.

omalizumab is a recombinant humanized monoclonal antibody that blocks the high - affinity fc receptor of ige. omalizumab has been approved for the treatment of moderate to severe asthma; however, there is currently more and more data showing promising in the management also of chronic urticaria. we present a case series of 19 patients with chronic urticaria treated in a university department with omalizumab and give an overview of the existing literature comprising an additional 59 cases as well as a total of 139 patients enrolled in two randomized controlled trials comparing omalizumab with placebo. the collective evidence points to omalizumab as a safe and effective treatment option for patients with chronic urticaria who do not sufficiently respond to standard therapy as recommended by existing guidelines.

post - laser - assisted in situ keratomileusis (lasik) ectasia (ple) consists of progressive corneal steepening with stromal thinning due to a reduction in biomechanical stability of the cornea.14 although ple is a rare adverse outcome of laser refractive surgery, it is a significant sight - threatening complication leading to compromised visual function and patient morbidity.14 corneal collagen cross - linking (cxl) halts progressive corneal ectasia by strengthening the collagen lamellae of the cornea.5 although this treatment was only recently approved by the us food and drug administration, cxl efficacy and safety have been extensively documented since 2003 in both keratoconus (kc) and ple patients.59 traditional epithelium - off cxl involves removal of the corneal epithelium, and therefore recovery is uncomfortable and long, with significant time off work, productivity loss, and compromised lifestyle. furthermore, the potential for significant complications exists.10 epithelium - on techniques have been developed to minimize recovery and complications, but have not shown the same efficacy and may not be suitable for ple.11,12 we propose a novel, less invasive technique to treat ple at an earlier clinical stage, before significant loss of cdva. the method we have termed under - flap cxl (ufcxl) involves soaking the stromal bed with riboflavin, repositioning the flap, then applying ultraviolet (uv) light to the corneal surface. we hypothesize that ufcxl will halt the ectatic progression in early ple by strengthening the corneal tissue under the flap and, in so doing, preserve existing refractive error, uncorrected distance visual acuity (udva), and corrected distance visual acuity (cdva) before it worsens. by eliminating the need to remove the epithelium, it can also minimize known complications of standard epi - off cxl, minimize patient discomfort, and significantly hasten recovery time. the purpose of this study is to describe this novel technique of ufcxl in treating early ple and to report on 6-month outcomes of the first treated eyes. inclusion criteria were early ple eyes, defined as presenting with new onset of progressive manifest refraction cylinder up to 1.50 d, udva of 20/40 or better, cdva of 20/25 or better, and new topographic irregular astigmatism and/or inferior steepening, with under - flap stromal bed of 325 m or greater, ing in a total corneal thickness (stromal bed + flap) of 400 m or greater. the study was approved by the ethics review board of the canadian ophthalmic research centre and fulfilled all principles of the declaration of helsinki. written informed consent was received from all patients. all procedures were formed using an internally - developed standardized technique for under - flap stromal cxl. the existing lasik flap was lifted and retracted back onto a weck pillow (merocel, beaver - visitec international inc ., waltham, ma, usa) on the superior bulbar conjunctiva. ultrasound pachymetry (sonogage inc ., cleveland, oh, usa) was performed on the stromal bed. a 7 mm circular sponge (corneal light shield, beaver - visitec international inc .) was soaked with drops of 0.25% riboflavin solution (riboflavin dissolved in 0.87% nacl ; haber s compounding pharmacy, toronto, on, canada) and was placed on the stromal bed for a duration of 3 minutes (similar to accelerated protocols used in adjunct lasik cxl1317), with meticulous care not to expose the flap to riboflavin. the quick riboflavin soak time, using a concentration of 0.25%, did not allow the retracted, exposed flap to dry out. the stromal bed was then dried, and any excess riboflavin was removed with a weck sponge. the flap was then refloated back into position, and remaining riboflavin was irrigated away. the flap was reexamined at the slit lamp and adjusted, if necessary, as per normal lasik procedure. accelerated uv light exposure was then performed as published elsewhere.1317 uv light (ccl - vario cross - linking radiation system ; peschke trade gmbh, huenenberg, switzerland) was applied for 3 minutes at 18 mw / cm, ing in a total irradiation dose of 3.24 j / cm at the corneal surface. after uv treatment, the flap was again verified to be free of striae, debris, and in good position. postoperative treatment of ufcxl eyes included the administration of zymar four times a day for 5 days, and pred - forte as follows: every hour on day 1; every 2 hours on day 2; and four times a day for days 3, 4 and 5. data from ophthalmic examinations at pre - lasik, pre - ufcxl, and 1, 2, 3, 6 months, and later time points post - ufcxl were collected for analysis, including manifest refraction sphere, cylinder, spherical equivalent (mrse), udva, cdva, maximum keratometry (kmax), central pachymetry, corneal irregularity indices, presence of haze, and/or other complications at slit lamp. keratometry and corneal irregularity indices were obtained with the orbscan iiz system (bausch & lomb, rochester, ny, usa) at each time point. all statistical analyses were carried out in matlab r2016b software (mathworks, natick, ma, usa) using two - sample paired student s t - tests (eg, to compare pre- and post - ufcxl variable means) and two - sample kolmogorov inclusion criteria were early ple eyes, defined as presenting with new onset of progressive manifest refraction cylinder up to 1.50 d, udva of 20/40 or better, cdva of 20/25 or better, and new topographic irregular astigmatism and/or inferior steepening, with under - flap stromal bed of 325 m or greater, ing in a total corneal thickness (stromal bed + flap) of 400 m or greater. the study was approved by the ethics review board of the canadian ophthalmic research centre and fulfilled all principles of the declaration of helsinki. written informed consent was received from all patients. all procedures were formed using an internally - developed standardized technique for under - flap stromal cxl. the existing lasik flap was lifted and retracted back onto a weck pillow (merocel, beaver - visitec international inc ., waltham, ma, usa) on the superior bulbar conjunctiva. cleveland, oh, usa ) was performed on the stromal bed. a 7 mm circular sponge (corneal light shield, beaver - visitec international inc .) was soaked with drops of 0.25% riboflavin solution (riboflavin dissolved in 0.87% nacl ; haber s compounding pharmacy, toronto, on, canada) and was placed on the stromal bed for a duration of 3 minutes (similar to accelerated protocols used in adjunct lasik cxl1317), with meticulous care not to expose the flap to riboflavin. the quick riboflavin soak time, using a concentration of 0.25%, did not allow the retracted, exposed flap to dry out. the stromal bed the flap was then refloated back into position, and remaining riboflavin was irrigated away. the flap was reexamined at the slit lamp and adjusted, if necessary, as per normal lasik procedure. accelerated uv light exposure was then performed as published elsewhere.1317 uv light (ccl - vario cross - linking radiation system ; peschke trade gmbh, huenenberg, switzerland) was applied for 3 minutes at 18 mw / cm, ing in a total irradiation dose of 3.24 j / cm at the corneal surface. after uv treatment, the flap was again verified to be free of striae, debris, and in good position. postoperative treatment of ufcxl eyes included the administration of zymar four times a day for 5 days, and pred - forte as follows: every hour on day 1; every 2 hours on day 2; and four times a day for days 3, 4 and 5. data from ophthalmic examinations at pre - lasik, pre - ufcxl, and 1, 2, 3, 6 months, and later time points post - ufcxl were collected for analysis, including manifest refraction sphere, cylinder, spherical equivalent (mrse), udva, cdva, maximum keratometry (kmax), central pachymetry, corneal irregularity indices, presence of haze, and/or other complications at slit lamp. keratometry and corneal irregularity indices were obtained with the orbscan iiz system (bausch & lomb, rochester, ny, usa) at each time point. all statistical analyses were carried out in matlab r2016b software (mathworks, natick, ma, usa) using two - sample paired student s t - tests (eg, to compare pre- and post - ufcxl variable means) and two - sample kolmogorov data were available for eight eyes of seven patients, who underwent previous hansatome microkeratome lasik and presented with early ple. the average age was 33.89.6 years, mean pre - ufcxl udva of 0.180.13 logmar (20/30), cdva of 0.040.04 logmar (20/201), and cylinder of 0.780.49 d. average time to ufcxl was 30.513.5 months post - lasik, with an average follow - up time post - ufcxl of 7.64.1 months. there was no significant difference (p=0.44) in accuracy pre- to post - ufcxl, with r values of 0.99 vs. 0.96, respectively (figure 1a). a total of 50%, 88% and 88% of ple eyes were respectively within 0.25 d, 0.50 d, and 1.00 d of intended correction pre - ufcxl (figure 1b), compared with 38%, 50%, and 75% post - ufcxl (p=0.70). moreover, 25%, 50%, and 63% of eyes were within 0.25 d, 0.50 d, and 1.00 d of intended plano cylinder pre - ufcxl (figure 2), compared to 13%, 25%, and 63% post - ufcxl (p=0.69), respectively. at the time of diagnosis, early ple ed in a decreased lasik efficacy index of 0.790.25 pre - ufcxl, which was unchanged post - ufcxl (0.830.31, p=0.76). there was a nonsignificant (p=0.68) increase in the percentage of eyes achieving cumulative snellen udva of 20/20 and 20/30 in post - ufcxl (25% and 88%, respectively) vs. pre - ufcxl (13% and 63%, respectively) compared with pre - lasik cdva (50% and 100%, respectively ; figure 3a). this was apparent when comparing the difference in snellen lines of pre - lasik cdva to pre - ufcxl udva (25% with three or more lines worse and 0% with one or more lines better) and pre - lasik cdva to post - ufcxl udva (12.5% with three or more lines worse and 25% with three or more lines better, figure 3b). cylinder vector analysis revealed a nonsignificant trend toward overcorrection post - ufcxl compared with pre - ufcxl (figure 4a, correction index : 1.100.67 vs. 0.830.33, respectively ; p=0.44) and an increased index of success (ios) post - ufcxl compared with pre - ufcxl (ios : 0.861.12 vs. 0.580.52, respectively ; p=0.45). for both pre- and post - ufcxl compared with pre - lasik, the overall angle of error was within 25 and + 25 in the majority of eyes (75% and 75%, respectively ; figure 4b). there was a slight nonsignificant (p=0.26) increase in cylinder magnitude 6 months post - ufcxl (1.090.76 d), compared with pre - ufcxl (0.780.49 d). the safety index was unchanged pre- to post - ufcxl (1.030.09 vs. 1.040.15, p=0.89). five eyes had the same snellen cdva post - ufcxl (figure 5a), whereas one eye lost one line and two eyes gained one line. the cumulative snellen cdva was unchanged (p=0.93) between pre - lasik, pre - ufcxl, and post - ufcxl (figure 5b). mrse was stable post - ufcxl at all time points examined (p=0.44 at 6 months, figure 6a), as was cylinder (p=0.26, not shown). corneal topography showed no evidence of ectasia progression over the follow - up time period. kmax was stable across all time points (p=0.94 at 6 months, figure 6b). furthermore, there was no significant difference pre- to post - ufcxl in the orbscan iiz corneal irregularity indices within both the central 3 mm (3.210.87 vs. 3.191.04, respectively ; p=0.52) and 5 mm corneal areas (3.360.72 vs. 3.310.70, respectively ; p=0.73). one eye had mild, nonvisually significant haze that had fully resolved by the last post - ufcxl visit, and one eye had epithelial ingrowth requiring removal, which obtained 100% efficacy and safety after healing. there was no significant difference (p=0.44) in accuracy pre- to post - ufcxl, with r values of 0.99 vs. 0.96, respectively (figure 1a). a total of 50%, 88% and 88% of ple eyes were respectively within 0.25 d, 0.50 d, and 1.00 d of intended correction pre - ufcxl (figure 1b), compared with 38%, 50%, and 75% post - ufcxl (p=0.70). moreover, 25%, 50%, and 63% of eyes were within 0.25 d, 0.50 d, and 1.00 d of intended plano cylinder pre - ufcxl (figure 2), compared to 13%, 25%, and 63% post - ufcxl (p=0.69), respectively. at the time of diagnosis, early ple ed in a decreased lasik efficacy index of 0.790.25 pre - ufcxl, which was unchanged post - ufcxl (0.830.31, p=0.76). there was a nonsignificant (p=0.68) increase in the percentage of eyes achieving cumulative snellen udva of 20/20 and 20/30 in post - ufcxl (25% and 88%, respectively) vs. pre - ufcxl (13% and 63%, respectively) compared with pre - lasik cdva (50% and 100%, respectively ; figure 3a). this was apparent when comparing the difference in snellen lines of pre - lasik cdva to pre - ufcxl udva (25% with three or more lines worse and 0% with one or more lines better) and pre - lasik cdva to post - ufcxl udva (12.5% with three or more lines worse and 25% with three or more lines better, figure 3b). cylinder vector analysis revealed a nonsignificant trend toward overcorrection post - ufcxl compared with pre - ufcxl (figure 4a, correction index : 1.100.67 vs. 0.830.33, respectively ; p=0.44) and an increased index of success (ios) post - ufcxl compared with pre - ufcxl (ios : 0.861.12 vs. 0.580.52, respectively ; p=0.45). for both pre- and post - ufcxl compared with pre - lasik, the overall angle of error was within 25 and + 25 in the majority of eyes (75% and 75%, respectively ; figure 4b). there was a slight nonsignificant (p=0.26) increase in cylinder magnitude 6 months post - ufcxl (1.090.76 d), compared with pre - ufcxl (0.780.49 d). the safety index was unchanged pre- to post - ufcxl (1.030.09 vs. 1.040.15, p=0.89). five eyes had the same snellen cdva post - ufcxl (figure 5a), whereas one eye lost one line and two eyes gained one line. the cumulative snellen cdva was unchanged (p=0.93) between pre - lasik, pre - ufcxl, and post - ufcxl (figure 5b). mrse was stable post - ufcxl at all time points examined (p=0.44 at 6 months, figure 6a), as was cylinder (p=0.26, not shown). corneal topography showed no evidence of ectasia progression over the follow - up time period. kmax was stable across all time points (p=0.94 at 6 months, figure 6b). furthermore, there was no significant difference pre- to post - ufcxl in the orbscan iiz corneal irregularity indices within both the central 3 mm (3.210.87 vs. 3.191.04, respectively ; p=0.52) and 5 mm corneal areas (3.360.72 vs. 3.310.70, respectively ; p=0.73). one eye had mild, nonvisually significant haze that had fully resolved by the last post - ufcxl visit, and one eye had epithelial ingrowth requiring removal, which obtained 100% efficacy and safety after healing. cxl performed under a lasik flap (sometimes referred to as intrastromal cxl and trade named lasik xtra by avedro, waltham, ma, usa) has been used as an adjunct with lasik surgery. its purpose has been prophylaxis against refractive regression in high myopia or hyperopia and to prevent ectasia in higher risk cases.1315,1722 the literature already documents that such ufcxl is safe1315,1722 with no vision loss or endothelial cell loss. to the best of our knowledge though , ufcxl has never been described as a way to prevent further progression of an early ectatic process in ple diagnosed patients. the reported incidence of ple is low, varying between 0.04% and 0.6% depending on the population being studied.2,3 given the large number of lasik procedures performed each year, even a low incidence of ple is significant and warrants efforts to reduce its occurrence and also to improve on current available treatments. in the current study, using ufcxl we present a novel, less invasive technique than traditional epi - off cxl to treat ple at an early clinical stage, before significant loss of cdva. we treated seven patients (eight eyes), with cxl under the lasik flap (ufcxl), who developed clear signs of early ple. we used the same parameters (riboflavin concentration and uv light dose) as previously described accelerated protocols for adjunct lasik cxl. this technique leaves an intact epithelium and therefore has the potential to minimize known complications of corneal surgery and those of epi - off cxl, such as delayed epithelial healing and slow recovery, keratitis, dry eye, haze, loss of visual acuity, induced refractive error, and more.2327 none of the eyes presented here experienced any of these complications, including any significant haze. it is a potential complication of flap re - lifting, and needs to be monitored and dealt with as with all lasik flap relifts. to our knowledge, the only report similar to this technique for early ple is the case of a 28-year - old female who presented 4 months post - lasik with unilateral inferior corneal steepening and decreased visual acuity.28 the one eye was treated with what was termed intrastromal cxl, and ended up asymptomatic with 20/20 acuity and no sign of disease progression on corneal topography after 1 year.28 in contrast to our study, the flap was only opened, and riboflavin was injected between the flap and the stroma bed. the flap was, therefore, also soaked in riboflavin and was cross - linked. in our case series, the flap was fully retracted, and riboflavin was administered only to the stromal bed, and after removal of any excess solution, the flap was repositioned. uv light was then administered to the surface of the cornea, thereby cross - linking the riboflavin - soaked stromal bed under the flap. this ufcxl technique allows strengthening and stiffening of the underlying cornea rather than the flap, which in lasik surgery provides little biomechanical strength to the cornea.29 our inclusion criteria were topographic and refractive signs consistent with early ple. in this way , any ectatic progression could be halted at a point where visual acuity was not yet significantly compromised, and good vision could be preserved after treatment. our preliminary demonstrate the maintenance of visual accuracy, efficacy, and safety at 6 months post - ufcxl, with quicker recovery times than traditional epi - off cxl. stability was also maintained at 6 months, with no evidence of progressive ectasia based on refraction, vision, kmax, topographic cylinder, and corneal irregularity indices. post - lasik patients are often not followed up routinely with regularly scheduled postoperative topographies. if they develop ectasia, they present with moderate to advanced disease once they complain of poor vision and seek an enhancement. as well, the few ple patients who do present early, with only topographic signs of ectasia yet minimal refractive changes and good udva and cdva, are often not immediately treated. both surgeons and patients are reluctant to treat (or be treated) with standard epi - off cxl at these earliest ple stages due to the risk of cxl complications, the potential for the procedure itself to worsen vision, the long recovery period, and the surgeon s reflex to document progression as with keratoconus, before embarking on a relatively invasive intervention.2327 as a , ple patients tend to be treated months to years after early ple signs, with their cdva already compromised.7,30,31 while it is indeed difficult to determine which new cases of early ple will be progressive and to what extent, and without a control group in this study, we can not be definitive that it is the intervention that halted progression, we do know that a large number of ple cases lead to significant visual loss with difficult rehabilitation affecting quality of life. we also know that a high percentage of kc patients progress. having a lasik flap that destabilizes a cornea may cause a greater percentage of ple patients to progress. a treatment that poses little risk to safety or visual downside allows immediate intervention once a diagnosis of ple is made, in order to preserve vision before significant loss of cdva occurs and before significant irregular astigmatism contributes to complicated visual rehabilitation. with regular monitoring, if progression were detected, ufcxl could be repeated or epi - off cxl initiated at that time. although the current follow - up was only 6 months (with one eye followed for 18 months), if stability maintains long term as with epi - off cxl treatment, then treating early ple eyes with ufcxl would outweigh the risks of disease progression followed by treating with the epi - off technique. it would also change the standard of care for post - lasik surgery, requiring consistent long - term follow - up with regularly scheduled topographies, looking for early signs of ectasia that could benefit from early ufcxl in order to preserve vision. while this preliminary experimental case series provides first data and guidelines for this novel ple treatment strategy, it has limitations. our small sample of patients (eight eyes) reflects the low incidence of ple and potentially limits our statistical power. the latter might have prevented us from detecting subtle trends in the data, such as the slight increase in post - ufcxl cylinder documented herein. in addition, we did not perform optical coherence tomography imaging to assess the depth of the cxl demarcation line, thought to indicate the extent of cxl effect. notwithstanding, we used the same protocols as with adjunct lasik cxl which showed good effect.1317 even smaller total irradiation doses demonstrated sufficient uv light absorption with post - treatment demarcation lines as deep as 280 m. considering that the clinical scenario in this study is ple with only under - flap tissue being cross linked, higher total doses may be required. the amount of total energy delivered to the stromal bed and the actual amount of cross - linked tissue necessary to halt progression in early ple needs to be elucidated with further studies, and reliable in vivo biomechanical measurements would prove useful. while in vivo confocal microscopy revealed no endothelial cell loss 12 months after adjunct cxl with lasik,13 endothelial cell count was not performed herein and should therefore be investigated in future ufcxl studies. our preliminary 6-month are promising, demonstrating maintenance of visual accuracy, efficacy, and safety, with no evidence of progressive corneal ectasia based on vision, refraction, kmax, topographic cylinder, and corneal irregularity indices. while our study is limited to a small sample of eyes, reflecting the low - incidence of ple, it suggests that ufcxl has the potential to be a safe and effective method of preserving visual function by halting ple progression early, prior to its detrimental effects. control studies with more patients and longer follow - up times are required to further validate these findings.

purposecollagen cross - linking (cxl) for post - laser - assisted in situ keratomileusis (lasik) ectasia (ple) is traditionally performed either epi - on or epi - off on the corneal surface. this study describes a novel technique in treating early ple with under - flap cxl (ufcxl) to the stromal bed and reports on 6-month outcomes.patients and methodscase series of seven patients (eight eyes) with topography - diagnosed early ple treated with ufcxl. inclusion criteria were early, mild ple defined as new - onset postoperative manifest refraction cylinder 1.50 d, with new topographic inferior steepening consistent with ectasia, uncorrected distance visual acuity (udva) of 20/40 or better, and corrected distance visual acuity (cdva) of 20/25 or better. existing lasik flap was lifted, riboflavin was applied directly to the stromal bed, flap was repositioned, and 18 mw / cm2 ultraviolet light was applied for 3 minutes to the corneal surface. post - ufcxl manifest refraction, udva and cdva, corneal cylinder, kmax, and corneal irregularity index were compared with pre - ufcxl measurements.patients had a pre - ufcxl sphere of 0.090.48 d and cylinder of 0.780.49 d. at 6 months, post - ufcxl sphere (0.060.8 d ; p=0.89) and cylinder (1.090.76 d, p=0.26) were unchanged. cumulative post - ufcxl udva was unchanged, achieving 20/20, 20/30, and 20/40 in 25%, 88%, and 88%, respectively, compared with 13%, 63%, and 88% pre - ufcxl (p=0.68). post - ufcxl cdva was unchanged (p=0.93) with a gain of one line in two eyes, a loss of one line in one eye, and five eyes unchanged. the efficacy index (p=0.76), safety index (p=0.89), kmax (p=0.94), and corneal irregularity index (p=0.73) were also unchanged.preliminary with ufcxl for early ple are promising, demonstrating maintenance of visual accuracy, efficacy, safety, kmax, and cylinder, with much quicker recovery times than surface cxl.

point - of - care (poc) is, essentially, the delivery of care wherever the patient may be and a point - of - care technology is defined as a technology that is used for bringing care to the patient rather than taking the patient to care. realizing the full potential of point - of - care technologies (poct) represents a critical factor in advancing overall health care by making the predictive, preemptive, preventive, and personalized care of the future accessible to all communities. to advance point - of - care, in 2006 the national institute of biomedical imaging and bioengineering (nibib) proposed a nationwide initiative to create a research network that would build multidisciplinary partnerships and expertise in the development of integrated systems that address unmet clinical needs at the point - of - care called the point - of - care technologies research network (poctrn). an april, 2006 workshop improving health care accessibility through point - of - care technologies sponsored by the nibib; national heart, lung, and blood institute (nhlbi); and the national science foundation (nsf) assessed the clinical needs and opportunities for use of poct in primary care, the home, emergency medical services, and in developing countries, and highlighted the state - of - science in sensors and microsystems, low - cost imaging, non - invasive monitoring, and telehealth / informatics. the creation of multidisciplinary partnerships was considered a key translational aspect for moving technologies from an early stage of development into clinical testing. since 2006 , the goal of the poctrn has been to develop technologies with clinical applications using a network model that enhances complementary strengths and builds multidisciplinary partnerships. to achieve this, each poctrn center funded by nibib performs or facilitates four core functions: collaborates with physical, biochemical, and computational scientists, and with engineers on exploratory technology development projects.completes clinical needs assessments in areas anticipated to advance the field of point - of - care testing and disseminates this information to the technology development community.provides training to technology developers on clinical issues related to the development of point - of - care devices.provides an adequate administrative structure to ensure that each large complex center achieves its goals. collaborates with physical, biochemical, and computational scientists, and with engineers on exploratory technology development projects. completes clinical needs assessments in areas anticipated to advance the field of point - of - care testing and disseminates this information to the technology development community. provides training to technology developers on clinical issues related to the development of point - of - care devices. provides an adequate administrative structure to ensure that each large complex center achieves its goals. the network also aims to drive the development of poct through collaborations that simultaneously merge scientific and technological capabilities with clinical need. these collaborative efforts include: utilizing clinical needs to direct technology development.leveraging expertise across centers to address a range of clinical needs and provide a systems focus.establishing partnerships to create a pipeline from clinical need to field testing of prototype devices.expanding the network to provide resources to the broader clinical and technology development communities. utilizing clinical needs to direct technology development. leveraging expertise across centers to address a range of clinical needs and provide a systems focus. establishing partnerships to create a pipeline from clinical need to field testing of prototype devices. expanding the network to provide resources to the broader clinical and technology development communities. an additional objective is to conduct parallel educational activities that advance evidence - based point - of - care practice in critical care, primary outreach and in low - resource environments, including global health care settings. the network measures its success through the variety of center activities: coordination of cross - center communication and collaboration to achieve network objectives.evaluation and guidance regarding funding of new technology development projects.informing members of individual center activities to leverage resources, expertise, and interests across the network.implementing mechanisms for center - to - center interactions.development of policies and procedures to be shared across centers.identification of common clinical needs.expertise to develop prototype systems.establishment of clinical and industrial partnerships.coordination of education and training programs and other shared practices.community outreach.communication of the network s role in advancing point - of - care testing.dissemination of clinical needs information to the technology development community.increased awareness of poct.outcome studies and parallel funding. evaluation and guidance regarding funding of new technology development projects. informing members of individual center activities to leverage resources, expertise, and interests across the network. implementing mechanisms for center - to - center interactions. outcome studies and parallel funding. each awarded center serves to identify clinical needs in point - of - care testing and communicate those needs to guide device design and appropriate clinical testing early in the development process. centers also provide resources to both technology development and clinical communities and coordinate activities across their respective areas in the network. solicitation to support specialized centers through a cooperative agreement (u54 mechanism) that covers a spectrum of multidisciplinary and translational research and development activities. the initial funded centers focused their activities on emerging neurotechnologies, disaster readiness, and advancing point - of - care diagnostics for global health. the point of care technology research center in primary care, managed by cimit (consortium for improving medicine with innovation and technology), is a center - without - walls for the rapid transformation of emerging poct into commercially viable, clinically focused solutions for improving primary health care. cimit was founded in 1998 and harnesses the power of multidisciplinary and multi - institutional collaboration to speed the translation of high - impact research into clinical practice and broader dissemination by commercially licensable opportunities. the specific aims of the point of care technology research center in primary care are to: apply cimit s innovation model to create clinically - driven point - of - care solutions that address critical areas of unmet need in primary health care.identify and assess unmet primary care needs and develop performance criteria where point - of - care technology solutions would have the highest impact.select and develop the most promising poct into proof - of - concept prototypes.test and evaluate prototype performance in simulated clinical environments and clinical living laboratories relative to clinical performance and implementation criteria.transition prototypes that meet performance specifications into commercially licensable or start - up company opportunities or work with companies to adapt their technologies for use in primary care.train and educate relevant stakeholders including clinicians, engineers, scientists, students and industry partners in the innovation process as it applies to meeting health care needs.disseminate lessons learned and best practices in innovation methodology and process both nationally and internationally in collaboration with other poctrn centers. apply cimit s innovation model to create clinically - driven point - of - care solutions that address critical areas of unmet need in primary health care. identify and assess unmet primary care needs and develop performance criteria where point - of - care technology solutions would have the highest impact. select and develop the most promising poct into proof - of - concept prototypes. test and evaluate prototype performance in simulated clinical environments and clinical living laboratories relative to clinical performance and implementation criteria. transition prototypes that meet performance specifications into commercially licensable or start - up company opportunities or work with companies to adapt their technologies for use in primary care. train and educate relevant stakeholders including clinicians, engineers, scientists, students and industry partners in the innovation process as it applies to meeting health care needs. disseminate lessons learned and best practices in innovation methodology and process both nationally and internationally in collaboration with other poctrn centers. primary care providers are at the frontlines of our health care system and serve as the first point of contact for care and as the referral pathway for specialty care. at the same time that the demand for care is increasing with an aging population and an increased burden of chronic disease, the pool of primary care providers is shrinking. this imbalance in supply and demand constrains primary care capacity, limiting access and fragmenting care as patients and families seek out other points of entry, often more expensive and less interconnected, into the health care system. the introduction of poct into primary care would increase the capacity of practices to care for more patients by eliminating inefficient testing turnaround delays and the need for post - visit communication of and recommendations to patients, freeing up clinical time. it is estimated that 55% of a typical primary care physician s day is spent outside of the examination room, primarily focused on follow - up and documentation of care for patients not physically present. this administrative rework is not only time - consuming, but discontinuous care jeopardizes patient outcomes in that the start of effective therapy may be delayed or, even worse, abnormal may not be communicated back to the primary care physician from the laboratory or to the patient by the physician, ing in a serious failure to diagnose because of lack of follow - up. for example, cimit has optimized its approach for maximizing potential clinical impact of innovations as a three - stage find, fund, and facilitate process that supports development of a pipeline of ideas from early stage proof - of - concept to clinical implementation and commercialization. to support project teams in navigating the healthcare innovation cycle (fig . 1), the center uses a variety of integrated methods and processes that are referred to collectively as the cimit model of innovation. finding significant unmet medical needs and identifying potential clinical collaborators is a critical ingredient in the innovation process and the starting point for the innovation journey. the center supports a needs assessment core managed by the massachusetts general hospital (mgh) john d. stoeckle center for primary care innovation. clinical involvement at all stages of the innovation process is key and so clinical collaborators must be an integral part of all projects funded by the center. funding is awarded on a competitive, peer - review basis once the need has been established. advancing to the next stage of the cycle, teams design potential solutions based on evaluating a range of technologies or by creating new ones; robust prototypes can then be developed and tested; and evidence is gathered to achieve regulatory approval. finally, to ensure success, the solution must be broadly disseminated so that it can become a new standard of care. the poctrc funds projects in three stages: 1 ) innovation stage (often called pilot, proof - of concept, or proof - of - principle), 2 ) acceleration stage (proof - of - value, often called validation), and 3 ) a clinical adoption / diffusion stage. the center circulates a call - for - proposals for innovation awards on an annual basis and typically funds 4 - 6 proposals. innovation awards are translational in nature and typically one year in duration, with budgets up to $ 100,000 (direct costs) with the objective of scientifically de - risking a novel idea by showing early - stage proof of concept, thereby justifying proceeding on to the proof - of - value stage to generate a viable candidate for further pre - commercial development. to date , the center has funded 27 innovation awards at different levels of maturity across the development pipeline. seven of these teams are developing multiplexed platform technologies for clinical laboratory testing from small blood samples with delivered within 15 minutes at the time of the primary care visit. projects with the potential for rapid transition to clinical implementation and hand - off to industry within 12 - 18 months are candidates for cimit s accelerator program and/or the program s healthcare communication course (both are described below). in addition to funding projects that are pre - commercial, the center also offers a clinical adoption and diffusion award to assist primary care practices in adopting and adapting commercially available poct to demonstrate value and speed adoption in the primary care setting. four accelerator awards and two clinical adoption and diffusion awards the percentage of awards to small companies has increased to represent approximately 80% of the award base consistent with the center s mission to rapidly advance innovations to impact patient care through commercialization. facilitating the journey through the healthcare innovation cycle is never smooth; many challenges are encountered at every point along the cycle. the center has assembled a team of experts in primary care and laboratory medicine complementing the strong cimit network of seasoned experts in translational research to address these challenges. late stage projects that are scientifically and technically de - risked and have well - understood market channels, but require further development and investment of money and/or business development expertise to attract interest from an entrepreneur or commercial entity to achieve initial market penetration, may be candidates for facilitation by mentors from the center s accelerator program, serial entrepreneurs who are former founders or ceos of medtech companies and who have an intimate understanding of the medical device and diagnostic markets. these seasoned executives join projects essentially full - time to drive them to commercial success by working with project teams to develop and execute a complete plan that includes a go - to - market strategy as well as market, competitive, financial and intellectual property analysis. funded teams may also participate in a 10-week healthcare commercialization craash (commercialization accelerator to advance solutions in healthcare) course, taught by industry veterans and based on decades of experience from the coulter foundation, massachusetts institute of technology (mit), yale, and cimit. the program formalizes development of a tested business model through the process of validating business hypotheses. teams receive 1:1 mentoring from successful healthcare entrepreneurs and group coaching from commercialization experts and investors. other core resources include cimit s virtual collaboration platform (colab), a systems engineering core, a clinical validation core, an education core and a medical device interoperability program. cimit colab the virtual collaboration platform is a synthesis of best - in - class, secure, cloud - based tools with functionality designed to allow users to manage the activities of the center, and to support teams through the healthcare innovation cycle. colab was used by the poctrn to develop a web portal for easy access to information common to all centers, including events, solicitations, clinical needs assessment, and prior awards. the systems engineering core was developed based upon the recognition that new poct do not exist in isolation; rather, they exist within ecosystems of other technologies and systems, and these systems influence their likelihood of success or failure and their effectiveness. nibib and center leadership convened a workshop in 2013 to explore the future of point of care testing and technologies. the clinical validation core provides a range of services, including clinical samples for testing, bench validation in a laboratory setting, and clinical trials in the mgh ambulatory practice of the future (apf), as well as other primary care settings. as an example, the apf in collaboration with the mgh clinical laboratories published the of a pilot study of implementation of a point - of - care testing satellite laboratory in a primary care practice. findings demonstrated high patient / provider satisfaction, increased efficiency, and financial viability of the model,. plug - and - play (md pnp) program, led by dr. julian goldman, is an important resource for the center relative to the definition of device requirements and performance standards. the program was established to lead the development and adoption of open standards and related technologies to facilitate widespread clinical use of medical device data and network - based medical device integration. the center for future technologies in cancer care (cftcc) is located at boston university (bu). the cftcc comprises several cores that work together to identify clinical needs, assess new technologies, build prototypes and educate stakeholders. the center focuses on a range of technologies for deployment along the continuum of care to improve early detection, enhance the lives of patients in care, monitor survivors, and support caretakers. the specific aims of the cftcc are to: enable and foster clinician / engineer partnerships.perform clinical needs assessments and impact analysis.fabricate alpha prototypes in the alpha prototyping core facility for center stakeholders.make fully functional prototypes for clinical testing in conjunction with the fraunhofer center for manufacturing innovation s (fcmi) beta prototyping facilities.provide training and networking opportunities for center stakeholders.promote poct for cancer nationally and internationally. make fully functional prototypes for clinical testing in conjunction with the fraunhofer center for manufacturing innovation s (fcmi) beta prototyping facilities. we have built an integrated multidisciplinary team consisting of engineers, clinicians, public health practitioners, and technology transfer experts. this team evaluates technologies in various stages of development for their suitability across a range of primary care and non - traditional healthcare settings. development, commercialization, and deployment of poc treatments for cancer has the potential to reduce healthcare costs by catching more cancers at earlier stages of progression, enabling treatment outside of specialized cancer centers, and monitoring at - risk patients remotely through mobile health strategies. moving cancer treatments out of specialized centers and into local clinics or home care could significantly lower healthcare costs in many settings. often patients have to travel long distances to receive treatments at cancer centers, or make repeated trips for monitoring over many months. in low - resource settings in the developing world surgical treatments carry infection risks, and in many places there are not enough surgeons to treat all of the patients in need. technologies like targeted ultrasound and light - based treatments could allow providers with less specialized training to treat more patients for less money. tools for monitoring chemotherapy patients at home between treatments could eliminate travel and office visits. mobile health strategies for collecting data about high - risk populations could lead to new interventions to directly impact cancer screening rates. the center focuses on the identification, prototyping, and early clinical assessment of innovative poct for the treatment, screening, diagnosis, and monitoring of a range of cancers. a major aspect of this effort involves assessing early stage technologies in terms of clinical needs, market demands, setting appropriateness, and commercialization strategies (fig . the center comprises an administrative core, a clinical needs assessment and impact analysis core, a training core, and a prototype development and testing core divided into two parts, the alpha and beta cores, which are both available to center stakeholders, depending on the stage of technology development . the team evaluates technologies in various stages of development for their suitability across a range of primary care and non - traditional healthcare settings . an important part of the poct design process is to move ideas from the lab bench into user - friendly formats . while presentations and drawings can be useful tools in communicating ideas, having a prototype device that a potential end user, manufacturer, or investor can hold in their hands is much more valuable . the alpha prototype in the design phase is a critical time to work out potential bugs or incompatibilities in the system before large scale investments are made in beta prototypes . the cftcc alpha core prototyping lab on the bu campus enables researchers ( from industry, academia, and medicine) from all across the country to build rapid prototypes of their devices. the alpha core houses basic equipment for the prototyping of microfluidic and paperfluidic devices. parts can be prototyped using standard photolithography and pdms processing with the assistance of core staff. core staff will also make and ship small (less than 10) batches of parts to investigators working on translating benchtop assays into microscale platforms. the core can laser cut, hot emboss, and heat seal thermoplastic devices with microscale features. other prototyping capabilities include xurography (cutter - plotter), wax printing, and reagent spotting on plastic and paper devices. finally, the core staff can work with developers of mobile applications to design user interfaces. the beta prototyping core is housed at the fraunhofer center for manufacturing innovation in brookline, ma, which is co - located with the bu campus. the beta core is tasked with fabricating prototypes that can be used in pre - clinical and feasibility testing. the design of these work - like prototypes is often iterative and involves clinical and engineering collaborators. the fcmi team has expertise in optical device design (endoscopic tools), microfluidic chip fabrication, assay development, and instrumentation. investigators with funded cftcc prototype projects are screened for fit with the beta core capabilities. often projects transition from the lab to the beta core after a year of center funding. the training core leads the cftcc efforts in outreach to the wider community of technology developers, researchers, clinicians, and patients and their advocates. programs include seminars, workshops, and trainings aimed at promoting poct and transfer of those technologies out of the lab. educating test and device developers about regulatory requirements, reimbursements, planning for human subjects research, and managing intellectual property concerns are missions of the core. all training activities are live streamed and archived. the clinical needs assessment (cna) core works in conjunction with the dana farber cancer institute to carry out and disseminate survey research aimed at determining where and when poct could make the most impact in patient care. over the last four years , the cftcc has funded 17 prototyping projects, three fellowships, and several small exploratory projects in our alpha prototyping core. the vast majority of these projects have secured follow on funding from federal and private sources. the reach of the center is national with projects at bu, the university of california davis, the university of california berkeley, massachusetts general hospital (mgh), the university of texas austin, michigan state university, the university of arizona, and the massachusetts institute of technology (mit). the cftcc conducts an open call for new projects in the fall of each year to fund 3 - 7 prototyping projects. after one year, if projects have met their proposed milestones, they are encouraged to apply for follow - on funding for specific translational activities. projects may also be referred to the beta core and receive in - kind services from that core to develop or enhance a prototype device. these projects include several point - of - care tests for the early detection of cancer or monitoring of disease , mobile health applications to coordinate care and patient outreach,, and devices and tools to enable minimally invasive testing at lower level care facilities including in primary care. the beta prototyping core has completed prototypes for researchers at the university of arizona, mit, bu, and for industry. prototypes include a wearable device for breast cancer chemotherapy monitoring, microfluidic sample preparation modules, and an instrument to fabricate hydrogel reagents for high - throughput point - of - care screening tests. a specific focus of the center is on identifying clinical needs in high disease burden populations and funding new technologies that address those needs. the cna core published a survey study conducted to determine the preferences for poct in cancer screening among primary care clinicians. the cna also worked with an international group of scientists and clinicians working on point - of - care devices to identify barriers to translation in low resource settings and, in particular, west africa. the training core has posted the assembled multimedia resources from all major outreach projects conducted during the past 4 years. the cftcc website serves as a valuable resource for new funding opportunities for cancer researchers interested in poct, and for test and device developers interested in applications in cancer care. we have hosted and co - hosted a semi - annual course called cancer care for engineers and scientists and workshops focusing on regulatory issues in addition to day - long science symposia. in 2016, the course new directions in cancer care for nonspecialists: immunotherapy and resistance to therapy was well attended both in person and virtually. this workshop was a full - day meeting for clinicians, engineers, and scientists to come together and learn about a very exciting new topic in cancer care. the alpha prototyping core hosts annual hack - a - thon events each october at the bu engineering product innovation center (epic). in addition to the competition, this event continues to raise awareness of the center and its resources in the field of cancer care technologies. the alpha prototyping core also participated in the camtech: global cancer innovation hack - a - thon in the spring of 2016, and winners of this event were awarded alpha core time to generate their first prototype units for testing. several of the hackathon participants are also working with the clinical needs assessment core to identify market opportunities for their new technologies. there are over 110 million prevalent stds / year and 19.7 million incident stds in the u.s./year, with new estimates of health care costs at $ 15.6 billion. costs for chlamydial infections are estimated at $ 516.7 million; gonorrhea, $ 162.1 million; hepatitis, $ 50.7 million; hiv, $ 12.6 billion; human papillomavirus, $ 1.7 billion, herpes simplex virus (hsv-2), $ 540 million; syphilis, $ 39.3 million; and trichomonas infections, $ 24.0 million. have great potential to significantly play a role in addressing the epidemics of stds in resource constrained and poor countries. stigma, privacy and confidentiality issues unique to std on top of issues of health inequity and health disparities in populations make stds optimal for poct, as well as home care tests or over - the - counter tests (otc). our mission is to drive the development of appropriate poct through collaborations that merge scientific and technological capabilities with clinical need. the long - term goals of our center are to address the epidemics of sexually transmitted diseases (stds) in the u.s. and in resource - poor settings by development and better use of poct, the center is organized in the following manner: 1.an administrative structure to ensure that the large, complex center achieves its goals in a timely manner. to ensure each functioning component works smoothly together and integrates with other centers in the nibib poctrn, our administrative component functions to accelerate a smooth transition along a pipeline from developmental prototype assays through in - house and pilot testing. in this coordinated manner , we can assist with the logical development of point - of - care assays that are proven to have sufficient scientific merit and can progress towards eventual clinical trials and fda submission.2.a clinical needs assessments and health impact assessments component for clinicians, end - users, and other appropriate stakeholders, in order to advance and inform the field of point - of - care testing design, development, and utilization by disseminating this information to the technology development community,.3.a collaboration component staffed with physical scientists, biochemical scientists, computational scientists, and systems engineers providing support for exploratory technology for prototype development projects and industry for testing of more mature poct in beta and pilot evaluations to insure a logical progression of assays along a pipeline from the concept stage to the clinical testing stage (fig . 4).4.a clinical evaluation component for very mature point - of - care assays by providing real world testing in clinics worldwide inclusive of resource - poor countries. in this way, we both enhance use of poct in these settings such as emergency departments, home testing, as well as advance end - user familiarity, clinical acceptance, and ultimately the use of such tests, ing in measureable improved health outcomes especially for those settings experiencing health disparities and inequities.5.a developer training component to provide training across all career levels about clinical and process issues as well as needs related to the development of point - of - care devices in an iterative fashion, as well as to provide training for scientists in resource - poor settings and education of relevant stakeholders on the development and potential impact of poct.6.a subject matter expertise review component emphasizing clinical drivers of technology progression for introducing new or expanding existing point - of - care std technologies, through design assistance (study, instrument, assay) and operational feedback iterated to permit spiral development and allow for project off ramps in a go no - go manner, such that we insure that deliverables for each project are met. center resources include: 1.diagnostic expertise and advice for developers of poct for stds, including guidance and the ability to provide clinical de - identified specimens in limited numbers to new developers.2.the ability to advertise solicitations for funding opportunities in the form of small grants for the development of poct for stds.3.conducting and providing needs assessments with clinicians and patients and follow - up training for industry for the sage development of poct for stds that can be accurate, timely, and simple.4.a laboratory facility to beta test point - of - care assays, using our archived de - identified clinical samples.5.the capacity to assist clinical sites in the collection and testing of preclinical trial assays in an early stage of maturity to guide their further refinement.6.access to testing sites in places outside the clinic, such as the internet, the emergency department, and foreign countries. figure 4.center pipeline. an administrative structure to ensure that the large, complex center achieves its goals in a timely manner. to ensure each functioning component works smoothly together and integrates with other centers in the nibib poctrn, our administrative component functions to accelerate a smooth transition along a pipeline from developmental prototype assays through in - house and pilot testing. in this coordinated manner , we can assist with the logical development of point - of - care assays that are proven to have sufficient scientific merit and can progress towards eventual clinical trials and fda submission. a clinical needs assessments and health impact assessments component for clinicians, end - users, and other appropriate stakeholders, in order to advance and inform the field of point - of - care testing design, development, and utilization by disseminating this information to the technology development community,. a collaboration component staffed with physical scientists, biochemical scientists, computational scientists, and systems engineers providing support for exploratory technology for prototype development projects and industry for testing of more mature poct in beta and pilot evaluations to insure a logical progression of assays along a pipeline from the concept stage to the clinical testing stage (fig . 4). a clinical evaluation component for very mature point - of - care assays by providing real world testing in clinics worldwide inclusive of resource - poor countries. in this way, we both enhance use of poct in these settings such as emergency departments, home testing, as well as advance end - user familiarity, clinical acceptance, and ultimately the use of such tests, ing in measureable improved health outcomes especially for those settings experiencing health disparities and inequities. a developer training component to provide training across all career levels about clinical and process issues as well as needs related to the development of point - of - care devices in an iterative fashion, as well as to provide training for scientists in resource - poor settings and education of relevant stakeholders on the development and potential impact of poct. a subject matter expertise review component emphasizing clinical drivers of technology progression for introducing new or expanding existing point - of - care std technologies, through design assistance (study, instrument, assay) and operational feedback iterated to permit spiral development and allow for project off ramps in a go no - go manner, such that we insure that deliverables for each project diagnostic expertise and advice for developers of poct for stds, including guidance and the ability to provide clinical de - identified specimens in limited numbers to new developers. the ability to advertise solicitations for funding opportunities in the form of small grants for the development of poct for stds. conducting and providing needs assessments with clinicians and patients and follow - up training for industry for the sage development of poct for stds that can be accurate, timely, and simple. a laboratory facility to beta test point - of - care assays, using our archived de - identified clinical samples. the capacity to assist clinical sites in the collection and testing of preclinical trial assays in an early stage of maturity to guide their further refinement. access to testing sites in places outside the clinic, such as the internet, the emergency department, and foreign countries. 1.emergency department- we have conducted studies in busy high - volume emergency department (ed) settings to evaluate performance and feasibility of integrating fda - approved poct for stds into clinical practice, with a focus on patient self - testing and use of tablet - based kiosks. we developed and optimized a kiosk - based platform for obtaining consent for hiv testing in the ed, and integrated that method into practice. we evaluated an fda - cleared point - of - care test for trichomonas vaginalis (tv), demonstrating high level of patient and provider acceptability of self - testing with tablet - guided facilitation, and showed excellent performance of the assay and high level of patient and provider concordance (98%), wherein the patient self - collected the vaginal sample, processed the sample following kiosk directions, and interpreted their own .2.needs assessments component- although development of poct has been evolving for decades, the belief of if you build it, they will use it has been a critical barrier to the commercialization and adoption of these technologies. we have conducted key informant interviews, focus group sessions among practitioners specializing in std diagnosis and testing, std poct researchers, laboratorians and developers, and patient end - users to determine what an ideal poct would look like with regard to setting, user requirements, device performance (sensitivity and specificity), and device usability in terms of complexity and time requirement. we have extended our needs to adolescent medicine clinicians, ob - gyns, hiv providers, and primary care clinicians practicing in fqhcs who are required by the new affordable care act to provide std screening, diagnosis and treatment services.3.training component- while new technologies have been moving laboratory science into ever more exciting and amazing methods of disease detection, these new technologies still require a high level of laboratory skill and expensive equipment and reagents. our training component is embedded in the std / hiv prevention training center at johns hopkins university (jhu). the training center has endeavored to educate std point - of - care technology stakeholders about issues associated with developing diagnostics that will be practical, affordable, cost - effective, and still profitable to the developers.4.development of poct within the center a.at the baltimore campus of the university of maryland, the center project with professor chris d. geddes is developing low cost, ultra rapid, sensitive assays for chlamydia trachomatis and neisseria gonorrhoeae, to test them clinically, and then to commercialize the technology using the microwave accelerated metal enhanced technology (mamef).b.at johns hopkins whitaker school of engineering, a team led by professor tza - huei wang and his jhu biomems group is developing a microfluidic device for delivering a low - cost and mobile nucleic acid amplification test (naat) for std detection. dr. dong jin shin and colleagues developed a bioanalytic platform based on the principles of droplet magnetofluidics for the detection of chlamydia trachomatis with performances comparable to standard - of - care naat tests.c.collaboration with the cincinnati children s site provides access to a population of adolescent patients seeking primary, urgent, and emergency care at a large, urban, tertiary care academic medical center. they have demonstrated the acceptability and effectiveness of self - testing for stds among women using a point - of - care std test; the benefits of women learning their std at the time of testing; and the acceptability of self - collected vaginal swabs from women for stds, which allows women greater access to self - testing. we demonstrated that using privacy shelters, as well as use of a health van is an acceptable setting to men and women to collect genital samples for std testing. poc testing ed in improved antibiotic stewardship.5.std testing outside the clinic via internet recruitment a.we have successfully translated our internet recruitment std screening program called iwtk into public health practice, which allows a participant to collect a sample at home and mail it to the center laboratory for testing with modernization of the site to be hipaa compliant and accessed through a secure login with password protection so that the participant can obtain his / her in a secure manner online.b.trichomonas home poc test. our website developers revised the iwtk site to allow female participants to access a link to the home test research study for trichomonas (tv). a questionnaire assessed acceptability and experience of the process for women participants. the accuracy of the home test was compared to the gold - standard mail - in molecular test for tv. one of the key findings was that over 80% of the participants would test themselves at home for tv if the rapid tv test were available over-the-counter.c.hiv home poc test- a new study has begun to increase hiv screening in adolescents, women and men with health inequity, in blacks, and especially in men who have sex with men (msm), by promoting direct outreach to persons through the internet and smart phones by a ) the ability to order a self hiv test for performance at home and b ) providing a coupon on the iwtk website for a free hiv test at participating clinics, based on participant zip code.6.std testing in resource - limited settings- given the significant burden of stds in sub - saharan africa (ssa), we established a site at the infectious diseases institute (idi) at makerere university college of health sciences in kampala, uganda. we have focused our efforts on syphilis detection particularly in pregnant women, who have a prevalence of syphilis from 1 to 8% across ssa. rapid treponemal lateral flow assays can be used to diagnose patients at the point - of - care. both the trinity and sd bioline tests met the who standard for accuracy (> 85% sensitivity ; > 95% specificity) against the sequential algorithm. the idi poc std site has also screened more than 15,000 pregnant women as part of a clinical trial to improve syphilis partner notification, testing, and treatment (nct02262390). emergency department- we have conducted studies in busy high - volume emergency department (ed) settings to evaluate performance and feasibility of integrating fda - approved poct for stds into clinical practice, with a focus on patient self - testing and use of tablet - based kiosks. we developed and optimized a kiosk - based platform for obtaining consent for hiv testing in the ed, and integrated that method into practice. we evaluated an fda - cleared point - of - care test for trichomonas vaginalis (tv), demonstrating high level of patient and provider acceptability of self - testing with tablet - guided facilitation, and showed excellent performance of the assay and high level of patient and provider concordance (98%), wherein the patient self - collected the vaginal sample, processed the sample following kiosk directions, and interpreted their own . needs assessments component- although development of poct has been evolving for decades, the belief of if you build it, they will use it has been a critical barrier to the commercialization and adoption of these technologies. we have conducted key informant interviews, focus group sessions among practitioners specializing in std diagnosis and testing, std poct researchers, laboratorians and developers, and patient end - users to determine what an ideal poct would look like with regard to setting, user requirements, device performance (sensitivity and specificity), and device usability in terms of complexity and time requirement. we have extended our needs to adolescent medicine clinicians, ob - gyns, hiv providers, and primary care clinicians practicing in fqhcs who are required by the new affordable care act to provide std screening, diagnosis and treatment services. training component- while new technologies have been moving laboratory science into ever more exciting and amazing methods of disease detection, these new technologies still require a high level of laboratory skill and expensive equipment and reagents. our training component is embedded in the std / hiv prevention training center at johns hopkins university (jhu). the training center has endeavored to educate std point - of - care technology stakeholders about issues associated with developing diagnostics that will be practical, affordable, cost - effective, and still profitable to the developers. development of poct within the center a.at the baltimore campus of the university of maryland, the center project with professor chris d. geddes is developing low cost, ultra rapid, sensitive assays for chlamydia trachomatis and neisseria gonorrhoeae, to test them clinically, and then to commercialize the technology using the microwave accelerated metal enhanced technology (mamef).b.at johns hopkins whitaker school of engineering, a team led by professor tza - huei wang and his jhu biomems group is developing a microfluidic device for delivering a low - cost and mobile nucleic acid amplification test (naat) for std detection. dr. dong jin shin and colleagues developed a bioanalytic platform based on the principles of droplet magnetofluidics for the detection of chlamydia trachomatis with performances comparable to standard - of - care naat tests.c.collaboration with the cincinnati children s site provides access to a population of adolescent patients seeking primary, urgent, and emergency care at a large, urban, tertiary care academic medical center. they have demonstrated the acceptability and effectiveness of self - testing for stds among women using a point - of - care std test; the benefits of women learning their std at the time of testing; and the acceptability of self - collected vaginal swabs from women for stds, which allows women greater access to self - testing. we demonstrated that using privacy shelters, as well as use of a health van is an acceptable setting to men and women to collect genital samples for std testing. poc testing ed in improved antibiotic stewardship. at the baltimore campus of the university of maryland, the center project with professor chris d. geddes is developing low cost, ultra rapid, sensitive assays for chlamydia trachomatis and neisseria gonorrhoeae, to test them clinically, and then to commercialize the technology using the microwave accelerated metal enhanced technology (mamef). at johns hopkins whitaker school of engineering, a team led by professor tza - huei wang and his jhu biomems group is developing a microfluidic device for delivering a low - cost and mobile nucleic acid amplification test (naat) for std detection. dr. dong jin shin and colleagues developed a bioanalytic platform based on the principles of droplet magnetofluidics for the detection of chlamydia trachomatis with performances comparable to standard - of - care naat tests. collaboration with the cincinnati children s site provides access to a population of adolescent patients seeking primary, urgent, and emergency care at a large, urban, tertiary care academic medical center. they have demonstrated the acceptability and effectiveness of self - testing for stds among women using a point - of - care std test; the benefits of women learning their std at the time of testing; and the acceptability of self - collected vaginal swabs from women for stds, which allows women greater access to self - testing. we demonstrated that using privacy shelters, as well as use of a health van is an acceptable setting to men and women to collect genital samples for std testing. std testing outside the clinic via internet recruitment a.we have successfully translated our internet recruitment std screening program called iwtk into public health practice, which allows a participant to collect a sample at home and mail it to the center laboratory for testing with modernization of the site to be hipaa compliant and accessed through a secure login with password protection so that the participant can obtain his / her in a secure manner online.b.trichomonas home poc test. our website developers revised the iwtk site to allow female participants to access a link to the home test research study for trichomonas (tv). the accuracy of the home test was compared to the gold - standard mail - in molecular test for tv. one of the key findings was that over 80% of the participants would test themselves at home for tv if the rapid tv test were available over-the-counter.c.hiv home poc test- a new study has begun to increase hiv screening in adolescents, women and men with health inequity, in blacks, and especially in men who have sex with men (msm), by promoting direct outreach to persons through the internet and smart phones by a ) the ability to order a self hiv test for performance at home and b ) providing a coupon on the iwtk website for a free hiv test at participating clinics, based on participant zip code. we have successfully translated our internet recruitment std screening program called iwtk into public health practice, which allows a participant to collect a sample at home and mail it to the center laboratory for testing with modernization of the site to be hipaa compliant and accessed through a secure login with password protection so that the participant can obtain his / her in a secure manner online. our website developers revised the iwtk site to allow female participants to access a link to the home test research study for trichomonas (tv). a questionnaire assessed acceptability and experience of the process for women participants. the accuracy of the home test was compared to the gold - standard mail - in molecular test for tv. one of the key findings was that over 80% of the participants would test themselves at home for tv if the rapid tv test were available over - the - counter. hiv home poc test- a new study has begun to increase hiv screening in adolescents, women and men with health inequity, in blacks, and especially in men who have sex with men (msm), by promoting direct outreach to persons through the internet and smart phones by a ) the ability to order a self hiv test for performance at home and b ) providing a coupon on the iwtk website for a free hiv test at participating clinics, based on participant zip code. std testing in resource - limited settings- given the significant burden of stds in sub - saharan africa (ssa), we established a site at the infectious diseases institute (idi) at makerere university college of health sciences in kampala, uganda. we have focused our efforts on syphilis detection particularly in pregnant women, who have a prevalence of syphilis from 1 to 8% across ssa. rapid treponemal lateral flow assays can be used to diagnose patients at the point - of - care. both the trinity and sd bioline tests met the who standard for accuracy (> 85% sensitivity ; > 95% specificity) against the sequential algorithm. the idi poc std site has also screened more than 15,000 pregnant women as part of a clinical trial to improve syphilis partner notification, testing, and treatment (nct02262390). finally, the technology exploration component (filling the development pipeline and integration and facilitation of technology development) works to seek out novel technologies and encourage their interaction with the center. up to three sub awards at $ 50,000 each are awarded per year from this component through open solicitations. the purpose of these tactical awards is to support key studies which will help them win larger awards toward commercial development of poct. solving many of today s unmet medical needs requires new models for successfully transforming healthcare. the point - of - care technologies research network represents a new partnership - based model that supports and facilitates collaborations across disciplines, institutions, and geographic regions to successfully drive innovative solutions from concept to patient care. the three centers within the network focus on commercially viable, point - of - care solutions for improving primary health care, the diagnosis and treatment of cancer, and the diagnosis of sexually transmitted diseases. as a network, centers provide resources to both technology development and clinical communities and coordinate activities with other centers. in this way, expertise, core resources, project support, and educational events are shared across centers, a process that is facilitated by frequent communication among center directors and program managers as well as the development of common policies and procedures across centers. built on the success of this nibib initiative, future trans - nih activities that focus on the advancement of multiplexed and multi - use poct will serve to enhance multidisciplinary partnerships and center - center synergies while supporting further development of integrated systems to address unmet health care needs.

to advance the development of point - of - care technology (poct), the national institute of biomedical imaging and bioengineering established the poct research network (poctrn), comprised of centers that emphasize multidisciplinary partnerships and close facilitation to move technologies from an early stage of development into clinical testing and patient use. this paper describes the poctrn and the three currently funded centers as examples of academic - based organizations that support collaborations across disciplines, institutions, and geographic regions to successfully drive innovative solutions from concept to patient care.

wegener's granulomatosis or granulomatosis with polyangiitis (gpa) is a multisystem autoimmune disease of unknown aetiology. its hallmark features include necrotizing granulomatous inflammation and pauci - immune vasculitis in small and medium - sized blood vessels of the respiratory tract and renal tract. gpa is one of the anti - neutrophil cytoplasmic antibody (anca)-associated vasculitides. the incidence and prevalence of gpa in the united kingdom is estimated at 10.2 and 250 cases per million population, respectively. gpa has a spectrum of clinical presentations and more than 75% of patients will develop renal disease. renal involvement manifests as a crescentic necrotizing glomerulonephritis, which may be associated with renal failure. current treatment recommendations in gpa depend on the severity and activity of disease, and include a combination of cyclophosphamide, glucocorticoids and in certain circumstances therapeutic plasma exchange. the rate of relapse after transplantation is approximately 0.09 per patient per year. in a study that ran from 1980 to 1995, the course of 13 patients with end - stage kidney disease (eskd) due to gpa was described. a post - kidney transplant relapse rate of 17.3% was derived by groups in sweden and the united states, combining the respective groups experience of 101 and 26 patients, respectively. renal involvement occurred in 12 of 22 relapse cases (55%) and ed in graft loss or declining function in 4 of 12. this equates to approximately an 18% relapse rate. in general, reports of relapse rates and severity of anca vasculitis following kidney transplant are low. here we describe two separate cases of gpa relapse in appropriately immunosuppressed renal transplant patients. our first case initially presented aged 14 in 2001 with a two - week history of vague ill health, back pain and intermittent haematuria. she was noted to have acute renal failure with both blood and protein in her urine; creatinine was 280 she was commenced on high - dose intravenous methylprednisolone for three days followed by oral prednisolone and pulsed intravenous cyclophosphamide once per month for three months. she required peritoneal dialysis for 34 weeks and was discharged, dialysis free with an estimated glomerular filtration rate of 24 ml / min/1.73 m. she remained on oral azathioprine as maintenance therapy. her anti - proteinase 3 (anti - pr3) level was < 5 iu / ml at the time of transplantation. she was well for the following seven years with a baseline creatinine of 12 mol / l, maintained on tacrolimus, azathioprine and prednisolone. in 2009 the patient was noted to have an increased creatinine of 200 mol / l, with arthralgia and right - sided episcleritis. her anti - pr3 antibody level was 26 iu / ml, increased from a baseline value of < 5 iu / ml. at this time she was on prednisolone 5 mg once per day, tacrolimus 2 mg twice per day and azathioprine 50 mg once per day. this showed features of pauci immune necrotizing and crescentic glomerulonephritis, consistent with a recurrence of gpa (fig . 1, fig . 2). her creatinine continued to rise, and her immunosuppression was further increased to include rituximab 1 g intravenously once per week for two weeks as treatment for relapse. it was thought that further courses of cyclophosphamide would not be appropriate, given her previous exposure to this drug, and given the risk of infertility. we also commenced a three - day course of intravenous methylprednisolone and changed her from azathioprine to mycophenolate. this patient did not achieve remission; her creatinine rose to 280 mol / l and her urine remained active. she was commenced on therapeutic plasma exchange, receiving a total of five four - litre exchanges over ten days. she was commenced on cyclophosphamide 15 mg / kg once per month for three months. this regime was based on a modified version of the euvas (european vasculitis study group) protocol. she achieved remission, and creatinine fell to 180 mol / l, with a normalisation of her urinary sediment. 3 ). a 64-year - old lady presented with eskd in 2001. her primary physician noted her to be hypertensive and to have a serum creatinine of 400 mol / l. perinuclear anca (panca) was positive with myeloperoxidase (mpo) specificity at a titre of > 700 iu / ml. the patient commenced haemodialysis three times per week, with a presumptive diagnosis of eskd secondary to panca - associated vasculitis. she did not receive any specific systemic treatment, as she did not have any features of active vasculitis. her anti - mpo antibody level was < 5 iu / ml at time of transplant. she was discharged on maintenance tacrolimus 1.5 mg b.d., and mycophenolate 500 mg b.d. this was based on emerging evidence at the time of the potential benefits of a steroid avoidance immunosuppressant regime. in 2009 mol / l to 200 mol / l in the setting of an unresolving lower respiratory tract process with pulmonary infiltrates. this was preceded by newly noted positive urinalysis, 3 + protein, 3 + blood. at this time she was on mycophenolate 250 mg b.d. and tacrolimus 0.5 mg b.d. her presentation was associated with panca positivity, with an anti - mpo antibody level of 640 iu / ml. a transplant biopsy was performed which revealed pauci immune necrotizing and crescentic glomerulonephritis. she was switched from mycophenolate to azathioprine, and was treated with pulsed intravenous methylprednisolone 1 g for three days. it was felt this patient's recurrence of gpa was due to her steroid - free immunosuppressive regime. she was switched from maintenance low - dose mycophenolate to azathioprine 100 mg / day; she was intolerant of higher doses of mycophenolate. our first case initially presented aged 14 in 2001 with a two - week history of vague ill health, back pain and intermittent haematuria. she was noted to have acute renal failure with both blood and protein in her urine; creatinine was 280 she was commenced on high - dose intravenous methylprednisolone for three days followed by oral prednisolone and pulsed intravenous cyclophosphamide once per month for three months. she required peritoneal dialysis for 34 weeks and was discharged, dialysis free with an estimated glomerular filtration rate of 24 ml / min/1.73 m. she remained on oral azathioprine as maintenance therapy. her anti - proteinase 3 (anti - pr3) level was < 5 iu / ml at the time of transplantation. she was well for the following seven years with a baseline creatinine of 12 mol / l, maintained on tacrolimus, azathioprine and prednisolone. in 2009 the patient was noted to have an increased creatinine of 200 mol / l, with arthralgia and right - sided episcleritis. her anti - pr3 antibody level was 26 iu / ml, increased from a baseline value of < 5 iu / ml. at this time she was on prednisolone 5 mg once per day, tacrolimus 2 mg twice per day and azathioprine 50 mg once per day. this showed features of pauci immune necrotizing and crescentic glomerulonephritis, consistent with a recurrence of gpa (fig . 1, fig . 2). her creatinine continued to rise, and her immunosuppression was further increased to include rituximab 1 g intravenously once per week for two weeks as treatment for relapse. it was thought that further courses of cyclophosphamide would not be appropriate, given her previous exposure to this drug, and given the risk of infertility. we also commenced a three - day course of intravenous methylprednisolone and changed her from azathioprine to mycophenolate. this patient did not achieve remission; her creatinine rose to 280 mol / l and her urine remained active. she was commenced on therapeutic plasma exchange, receiving a total of five four - litre exchanges over ten days. she was commenced on cyclophosphamide 15 mg / kg once per month for three months. this regime was based on a modified version of the euvas (european vasculitis study group) protocol. she achieved remission, and creatinine fell to 180 mol / l, with a normalisation of her urinary sediment. her primary physician noted her to be hypertensive and to have a serum creatinine of 400 mol / l. perinuclear anca (panca) was positive with myeloperoxidase (mpo) specificity at a titre of > 700 iu / ml. the patient commenced haemodialysis three times per week, with a presumptive diagnosis of eskd secondary to panca - associated vasculitis. she did not receive any specific systemic treatment, as she did not have any features of active vasculitis. her anti - mpo antibody level was < 5 iu / ml at time of transplant. she was discharged on maintenance tacrolimus 1.5 mg b.d., and mycophenolate 500 mg b.d. this was based on emerging evidence at the time of the potential benefits of a steroid avoidance immunosuppressant regime. in mol / l to 200 mol / l in the setting of an unresolving lower respiratory tract process with pulmonary infiltrates. this was preceded by newly noted positive urinalysis, 3 + protein, 3 + blood. at this time she was on mycophenolate 250 mg b.d. and tacrolimus 0.5 mg b.d. her presentation was associated with panca positivity, with an anti - mpo antibody level of 640 iu / ml. a transplant biopsy was performed which revealed pauci immune necrotizing and crescentic glomerulonephritis. she was switched from mycophenolate to azathioprine, and was treated with pulsed intravenous methylprednisolone 1 g for three days. it was felt this patient's recurrence of gpa was due to her steroid - free immunosuppressive regime. she was switched from maintenance low - dose mycophenolate to azathioprine 100 mg / day; she was intolerant of higher doses of mycophenolate. these cases describe the rare recurrence of anca vasculitis / gpa in appropriately immunosuppressed transplant patients. in previous studies , it was found that graft outcomes were more favourable if transplantation occurred at least a year post - induction of remission. it was also found that the presence of circulating anca at the time of transplantation was associated with increased severity of vasculopathy in subsequent transplant biopsies. the cases described above did not have these high - risk features. at the time of transplant , both had undetectable levels of anca, and both were transplanted at least one year after remission. this study also found that graft survival in those with an underlying diagnosis of anca - associated vasculitis was similar to graft survival in those with other causes of eskd. relapse of anca vasculitis in non - transplant patients was noted to be more common in patients treated with mycophenolate than in patients treated with azathioprine. although we can not directly extrapolate the findings of the improve trial to transplant patients, it does raise the possibility that patients with eskd secondary to anca vasculitis should be maintained on azathioprine instead of mycophenolate. the two cases described above emphasise the importance of regular follow - up of transplant patients, and informs us that although recurrence of anca vasculitis in transplant patients is rare, it should remain on our list of differential diagnoses in allograft disfunction.

granulomatosis with polyangiitis (gpa) (formerly known as wegener 's granulomatosis) is a multisystem autoimmune disease of unknown aetiology. renal disease manifests as a crescentic glomerulonephritis, with varying degrees of renal failure. ten percent of patients progress to end - stage kidney disease. relapse of gpa in renal transplant patients is rare, with a rate of 0.09 relapses per patient per year.patients and methodswe describe two cases of gpa relapse in immunosuppressed renal transplant patients.these patients presented with new - onset graft disfunction, having previously had an uncomplicated posttransplant course. both patients were on appropriate doses of immunosuppressive agents at the time of relapse, with therapeutic target levels of tacrolimus. we describe the history and management of both patients.the cases described inform us that although recurrence of anti - neutrophil cytoplasmic antibody vasculitis in transplant patients is rare, it should remain on our list of differential diagnoses in allograft disfunction.

leptomeningeal carcinomatosis (lmc) is defined as a diffuse or multifocal malignant infiltration of the pia matter and arachnoid membrane. it is clinically diagnosed in 510% of all cancer patients. the most commonly reported cancers associated with lmc are breast, lung, and hematological malignancies. symptoms are related to increased intracranial pressure, hemispheric dysfunction, cranial neuropathies, and spinal roots dysfunction. we report a case of lmc secondary to gastroesophageal junction cancer initially presenting with cauda equine syndrome. we present the case of a 51-year - old right - handed male patient diagnosed with signet ring adenocarcinoma of the gastroesophageal junction. nine months later, he complained of left hip pain followed by mild left leg weakness. subsequently, the patient was admitted with intestinal obstruction. during admission, he had gradual worsening of the left leg weakness and the development of right leg weakness. eight days following discharge, he presented to the emergency department with fecal incontinence and urinary retention requiring foley catheter insertion and worsening of his lower limb weakness. two days later, he developed progressive bilateral facial weakness, slurring of speech, and swallowing difficulties. his cranial nerve examination revealed facial diplegia, bilateral sensorineural hearing loss, and weak gag reflex. he showed hypotonia of lower limbs, severe asymmetrical weakness, and absent lower limb reflexes. 1 ) which showed diffuse leptomeningeal enhancement surrounding the spinal cord and cauda equina and associated with extension of the enhancement of the nerve roots. 2 ) showed diffuse leptomeningeal enhancement most prominent at basal csf cistern and cerebellum with extension into the cerebellar sulci and surround the brain stem. cerebrospinal fluid (csf) examination showed: protein = 648.3 mg / dl (1545 mg / dl), glucose = 7 mg / dl (4075 mg / dl), wbc = 39 cells / dl (lymphocytes 5970%), and rbc = 1124 cells / dl. repeat csf cytology showed malignant cells which is large atypical cells with enlarged nucleus and prominent nucleoli (fig . 3). immunohistochemistry the atypical cells are weakly positive for ck ae1/ae3 and negative for cd68. he had progressive worsening of his cranial neuropathies and died 10 days after hospital admission. the most common encountered malignancies are breast 35%, lung 24%, and hematological malignancies 16%. the development of lmc with gastrointestinal tumors is rare and most commonly reported with gastric cancer. few case reports described the development of lmc with esophageal cancer and gastroesophageal junction tumors. the tumor cells disseminated to the leptomeninges occur via direct invasion, hematogenous spread, or perineural spread especially in head and neck cancer. patients may present with features of increased intracranial pressure related to csf flow obstruction, neuronal dysfunction from metabolic competition for nutrients, and vascular alteration due to tumor growth. symptoms can be divided into those ing from hemispheric dysfunction, cerebellar dysfunction, cranial neuropathies, and spinal radiculopathies. in a study of 118 breast cancer patients with lmc, headache was the most common reported symptom (54%), followed by cranial neuropathies (42%), cerebellar signs (35%), paresis (26%), mental changes (19%), meningism (11%), seizure (9%), and radicular pain (7%). the development of cauda equina syndrome secondary to lmc has been reported previously in a 66-year - old female patient with advanced ovarian cancer. our patient normal imaging initially but subsequently it showed an evidence of diffuse leptomeningeal enhancement. high clinical suspicion should be considered in any cancer patient presenting with radicular symptoms and features of cauda equina syndrome. the presence of normal imaging studies does not rule out the presence of lmc. in the breast cancer lmc study by niwinska et al. initial csf examination will be positive only in 50% of cases, but the yield will increase up to 90% after the third exam. in cancer patients with cauda equina syndrome and absence of structural lesions on imaging, lmc should be considered and further testing with study of the csf is required. to our knowledge, this is the first case of lmc secondary to gastroesophageal cancer presenting with cauda equina syndrome.

leptomeningeal carcinomatosis (lmc) is a diffuse or multifocal malignant infiltration of the pia matter and arachnoid membrane. the most commonly reported cancers associated with lmc are breast, lung, and hematological malignancies. patients with lmc commonly present with multifocal neurological symptoms. we report a case of lmc secondary to gastroesopha - geal junction cancer present initially with cauda equina syndrome. a 51-year - old male patient with treated adenocarcinoma of the gastroesophageal junction presented with left leg pain, mild weakness, and saddle area numbness. initial radiological examinations were unremarkable. subsequently, he had worsening of his leg weakness, fecal incontinence, and urine retention. two days later, he developed rapidly progressive cranial neuropathies including facial diplegia, sensorineural hearing loss, dysarthria, and dysphagia. mri with and without contrast showed diffuse enhancement of leptomeninges surrounding the brain, spinal cord, and cauda equina extending to the nerve roots. cerebrospinal fluid cytology was positive for malignant cells. the patient died within 10 days from the second presentation. in cancer patients with cauda equina syndrome and absence of structural lesion on imaging , lmc should be considered. to our knowledge, this is the first case of lmc secondary to gastroesophageal cancer presenting with cauda equina syndrome.

muscle biopsies (50100 mg) were obtained from the vastus lateralis of lean (bmi 22.4 0.9 kg / m ; n = 12) and extremely obese (bmi 45.3 1.4 kg / m ; n = 9) women with the percutaneous needle biopsy technique. satellite cells were isolated and cultured into myoblasts as previously described. after reaching 70% confluency, cells were subcultured to examine the recombinant adenoviral overexpression of pgc-1 on fao, markers of mitochondrial content, and lipid accumulation as described below. recombinant adenoviruses encoding mouse pgc-1 (ad - pgc-1) or -galactosidase (ad--gal) were constructed, amplified, and purified as described previously. myoblasts were subcultured onto 6- and 24-well type i collagen - coated plates at densities of 80 and 20 10 cells per well, respectively. upon reaching 7080% confluence, differentiation to myotubes was induced by switching the growth media to differentiation media (dulbecco 's modified eeagle 's medium supplemented with 2% horse serum, 0.5 mg / ml bsa, 0.5 mg / ml fetuin, and 50 g / ml gentamicin / amphotericin b). on day five, myotubes were given fresh differentiation media (no - virus control) or transfected with either ad - pgc-1 or ad--gal (control virus). to determine the appropriate adenoviral titer to use for metabolic experiments , we initially performed a protein (fig . 1) and mrna (not shown) dose - response curve. ad - pgc-1 dose (5 10 plaque - forming units / ml) was used to mimic the effects of endurance exercise (3.5-fold increase in pgc-1 protein over controls) and a high ad - pgc-1 dose (1 10 plaque - forming units / ml) used to represent a supraphysiological increase in pgc-1 (approximately eightfold increase in pgc-1 protein over control). twenty - four hours after transfection, the medium was removed and replaced with fresh differentiation media. myotubes were harvested for respective experiments on day eight based on previous research. there were no obvious differences in the extent of myotube differentiation between lean and obese hskmc. on day eight of differentiation, myotubes were incubated at 37c in sealed 24-well plates containing differentiation media, 12.5 mmol / l hepes, 0.5% bsa, 1 mmol / l carnitine, either 100 mol / l or 500 mol / l sodium oleate (sigma - aldrich, st . louis, mo), and 1 ci / ml oleate (perkinelmer, ma) for 3 h. following incubation, medium was assayed for co2 (measure of complete oxidation), and radiolabeled acid soluble metabolites (asm) (measure of incomplete oxidation) as previously described. cells were washed twice with pbs, harvested in 600 l of 0.05% sds lysis buffer, and stored at 80c for subsequent determination of protein concentration and lipid esterfication. for determination of lipid esterfication , 500 l cell lysate was added to 1:2 chloroform: methanol (vol / vol). after vortexing, 625 l chloroform was added, followed by the addition of 625 l deionized h2o. the chloroform phase (containing total lipids extracted) was transferred to a clean glass tube and evaporated under a stream of 100% n2. samples were resuspended in 500 l of 2:1 chloroform: methanol. for the quantification of total lipids, 50 l of the sample 50 l of each sample was spotted onto oven - dried silica plates (silica gel gf ; analtech, newark, de), and placed in a sealed tank containing solvent (60:40:3 heptane : isopropyl ether : acetic acid) for 45 min. plates were air - dried and scanned for the visualization of the bands representing triacylglycerol (tag), diacylglycerol (dag), and phospholipid (pl). cells were washed twice with pbs and trypsinized with trypsin - edta (0.05% trypsin and 0.02% edta). total dna (mitochondrial and nuclear) was extracted from cells using a qiaamp dna minikit (qiagen, valencia, ca) and total dna quantified using the picogreen dna quantification kit (molecular probes, eugene, or). mtdna content was measured as relative copy number of mtdna per diploid nuclear genome using real - time pcr. , detection of a 69-bp fragment of mtdna (nucleotides 1491814986) and a 77-bp fragment of -globin were used as markers of mtdna and nuclear dna, respectively. primer and probe sets were purchased from applied biosystems (foster city, ca) using sequences previously reported by menshikova et al. real - time pcr was conducted using an abi prism 7900ht sequence detection system under conditions previously described. the threshold cycle number (ct) mtdna was expressed as a relative copy number (rc) by expressing ct differences between -globin and mtdna as previously described and based on the following calculations: rc = 2 and ct = ct-globin ctmtdna. cells were washed twice with ice - cold pbs and harvested in 150 l lysis buffer (50 mmol / l hepes , 1% triton x-100, 10 mmol / l edta, 100 mmol / l nafl, and 12 mmol / l na pyrophosphate) supplemented with protease and phosphatate inhibitors (sigma - aldrich). protein concentrations were determined from cell extracts using the bicinchoninic acid assay (pierce biotechnology, rockford, il). thirty micrograms of cellular protein were separated by sds - page and electrotransferred onto polyvinylidene difluoride membranes (millipore, billerica, ma) and probed overnight for either pgc-1 (1:500 ; cell signaling, beverly, ma), mitochondrial transcription factor (mttfa) (1:500 ; santa cruz biotechnologies, santa cruz, ca), or cytochrome c oxidase (cox)iv (1:8,000 ; cell signaling). comparisons between hskmc from lean and obese donors were performed with repeated - measures anova. significant main effects and interactions were further analyzed using contrast - contrast tests when appropriate. statistical significance was defined as a p value < 0.05, and data are presented as means se. recombinant adenoviruses encoding mouse pgc-1 (ad - pgc-1) or -galactosidase (ad--gal) were constructed, amplified, and purified as described previously. myoblasts were subcultured onto 6- and 24-well type i collagen - coated plates at densities of 80 and 20 10 cells per well, respectively. upon reaching 7080% confluence, differentiation to myotubes was induced by switching the growth media to differentiation media (dulbecco 's modified eeagle 's medium supplemented with 2% horse serum, 0.5 mg / ml bsa, 0.5 mg / ml fetuin, and 50 g / ml gentamicin / amphotericin b). on day five, myotubes were given fresh differentiation media (no - virus control) or transfected with either ad - pgc-1 or ad--gal (control virus). to determine the appropriate adenoviral titer to use for metabolic experiments, we initially performed a protein (fig . 1) and ad - pgc-1 dose (5 10 plaque - forming units / ml) was used to mimic the effects of endurance exercise (3.5-fold increase in pgc-1 protein over controls) and a high ad - pgc-1 dose (1 10 plaque - forming units / ml) used to represent a supraphysiological increase in pgc-1 (approximately eightfold increase in pgc-1 protein over control). twenty - four hours after transfection, the medium was removed and replaced with fresh differentiation media. myotubes were harvested for respective experiments on day eight based on previous research. there were no obvious differences in the extent of myotube differentiation between lean and obese hskmc. on day eight of differentiation, myotubes were incubated at 37c in sealed 24-well plates containing differentiation media, 12.5 mmol / l hepes, 0.5% bsa, 1 mmol / l carnitine, either 100 mol / l or 500 mol / l sodium oleate (sigma - aldrich, st . louis, mo), and 1 ci / ml oleate (perkinelmer, ma) for 3 h. following incubation, medium was assayed for co2 (measure of complete oxidation), and radiolabeled acid soluble metabolites (asm) (measure of incomplete oxidation) as previously described. cells were washed twice with pbs, harvested in 600 l of 0.05% sds lysis buffer, and stored at 80c for subsequent determination of protein concentration and lipid esterfication. for determination of lipid esterfication , 500 l cell lysate was added to 1:2 chloroform: methanol (vol / vol). after vortexing, 625 l chloroform was added, followed by the addition of 625 l deionized h2o. the chloroform phase (containing total lipids extracted) was transferred to a clean glass tube and evaporated under a stream of 100% n2. samples were resuspended in 500 l of 2:1 chloroform: methanol. for the quantification of total lipids, 50 l of the sample 50 l of each sample was spotted onto oven - dried silica plates (silica gel gf ; analtech, newark, de), and placed in a sealed tank containing solvent (60:40:3 heptane : isopropyl ether : acetic acid) for 45 min. plates were air - dried and scanned for the visualization of the bands representing triacylglycerol (tag), diacylglycerol (dag), and phospholipid (pl). cells were washed twice with pbs and trypsinized with trypsin - edta (0.05% trypsin and 0.02% edta). total dna (mitochondrial and nuclear) was extracted from cells using a qiaamp dna minikit (qiagen, valencia, ca) and total dna quantified using the picogreen dna quantification kit (molecular probes, eugene, or). mtdna content was measured as relative copy number of mtdna per diploid nuclear genome using real - time pcr. as recommended by miller et al. , detection of a 69-bp fragment of mtdna (nucleotides 1491814986) and a 77-bp fragment of -globin were used as markers of mtdna and nuclear dna, respectively. primer and probe sets were purchased from applied biosystems (foster city, ca) using sequences previously reported by menshikova et al. real - time pcr was conducted using an abi prism 7900ht sequence detection system under conditions previously described. the threshold cycle number (ct) mtdna was expressed as a relative copy number (rc) by expressing ct differences between -globin and mtdna as previously described and based on the following calculations: rc = 2 and ct = ct-globin ctmtdna. cells were washed twice with ice - cold pbs and harvested in 150 l lysis buffer (50 mmol / l hepes , 1% triton x-100, 10 mmol / l edta, 100 mmol / l nafl, and 12 mmol / l na pyrophosphate) supplemented with protease and phosphatate inhibitors (sigma - aldrich). protein concentrations were determined from cell extracts using the bicinchoninic acid assay (pierce biotechnology, rockford, il). thirty micrograms of cellular protein were separated by sds - page and electrotransferred onto polyvinylidene difluoride membranes (millipore, billerica, ma) and probed overnight for either pgc-1 (1:500 ; cell signaling, beverly, ma), mitochondrial transcription factor (mttfa) (1:500 ; santa cruz biotechnologies, santa cruz, ca), or cytochrome c oxidase (cox)iv (1:8,000 ; cell signaling). comparisons between hskmc from lean and obese donors were performed with repeated - measures anova. significant main effects and interactions were further analyzed using contrast - contrast tests when appropriate. statistical significance was defined as a p value < 0.05, and data are presented as means se. findings from the dose - response experiment for ad - pgc-1 are presented in fig. 1. treatment of myotubes with ad--gal (control virus) had no effect on pgc-1 protein content compared with that on no - virus controls (fig . 1a). in hskmc derived from lean and obese individuals, pgc-1 protein (fig . 1a) and mrna (data not shown) increased in a dose - dependent manner with increasing pgc-1 adenovirus titer; there were no differences between pgc-1 protein content in hskmc from lean and obese individuals with or without overexpression. 1a, for subsequent experiments we selected the 1-ul dose (5 10 plaque - forming units / ml) as pgc-1 protein content was increased by a magnitude similar to that reported with endurance - oriented exercise training (3.5-fold increase in pgc-1 protein over control) and the 2-l dose (1 10 plaque - forming units / ml) to represent a supraphysiological increment (approximately eightfold increase in pgc-1 protein over controls) in protein content. protein levels of mttfa and coxiv, indicators of mitochondrial biogenesis, also increased in a dose - dependent manner with pgc-1 adenovirus (fig . ad - pgc-1 overexpression in cultured myotubes ( hskmc) from lean and obese donors. a: pgc-1 protein content in no - virus controls (nvc), ad--gal controls, and ad - pgc-1treated hskmc. b: mttfa and coxiv protein content increased dose dependently with increasing pgc-1 viral titer in hskmc. to evaluate the role of pgc-1 in regulating skeletal muscle lipid oxidation, we treated myotubes from lean and obese donors with either low or high ad - pgc-1 for 24 h, followed by 3 h incubation with either 100 or 500 mol / l oleate. in all experiments, fao from the no - virus controls did not differ from that in the ad--gal controls (data not shown). 2. under control conditions (low and high -gal), complete fao (co2 production) was consistently depressed by 30% in hskmc from obese individuals with either the 100 or 500 mol / l oleate incubation (fig . 2a, overexpression of pgc-1 by 3.5-fold ( low dose) ed in an 30% increase in complete fao (100 mol / l oleate) in hskmc derived from lean and obese donors. the eightfold increase in pgc-1 protein (high dose) also increased complete fao in cells from both lean (64%) and obese (70%) donors at the 100 mol / l oleate concentration (p < 0.05) (fig . although pgc-1 overexpression increased fao regardless of the category of the donor, absolute values for complete fao remained depressed in hskmc derived from obese compared with those in lean subjects under all conditions ( fig . similar findings were obtained in response to the higher oleate concentration ( 500 mol / l) with respect to differences between cell types and the effects of pgc-1 overexpression (fig . total fao, determined as the sum of incomplete ( asm) and complete oxidation did not differ between cell type under any conditions (fig . 2c and d), and pgc-1 overexpression increased total oxidation in a dose - dependent manner. total and complete fao were significantly higher at 500 mol / l oleate compared with 100 mol / l oleate in control and pgc-1treated cells (p < 0.05) (fig . d). effect of pgc-1 overexpression on fao and oxidation efficiency in hskmc from lean and obese donors. hskmc cultured from lean (n = 12) and obese (n = 9) donors was treated with either low- or high - dose recombinant ad--gal or pgc-1 and incubated with either 100 mol / l (a, c, and e) or 500 (b, d, and f) mol / l oleate. complete fao was measured from c - labeled incorporation into co2 (a and b). total fao (c and d) was measured as the sum of c - labeled incorporation into co2 and c - labeled incorporation into asms, with asm serving as an index of incomplete fao. oxidation efficiency was determined as the ratio of asm to complete fao, represented as asm / co2 (e and f), with higher values indicative of reduced efficiency. data are expressed as means se and significant differences denoted at the p 0.05 level. * significant difference between lean and obese for that treatment. significant main effect comparing control (-gal) and pgc-1 overexpression (ad - pgc-1) at the respective adenoviral doses. significant increase in lean subjects with pgc-1 overexpression compared with control at the respective adenoviral doses. a ratio of incomplete (asm) to complete (co2) oleate oxidation was calculated as an index of fao efficiency. in control cells treated with 100 mol / l oleate, radiolabel incorporation into asm relative to co2 was approximately twofold higher in myotubes from obese compared with lean individuals (p < 0.05) (fig . the high relative rate of incomplete oxidation in the obese compared with the lean group was retained regardless of the level of pgc-1 overexpression or oleate concentration ( fig . pgc-1 overexpression increased the asm - to - co2 ratio by 55% ( p < 0.05); pgc-1 overexpression in cells from obese subjects had no effect on this ratio (fig . the asm / co2 ratio was significantly higher at 500 mol / l oleate compared with 100 mol / l oleate in control and pgc-1treated cells ( p < 0.05) (fig . control cells ( -gal) from obese individuals had a greater rate of oleate incorporation into the glycerolipid pool in response to low (64%) and high (42%) oleate concentrations (fig . 3a and b). cells from obese individuals, regardless of treatment, consistently incorporated more lipids into the glycerolipid pool compared with hskmc from lean subjects (fig . pgc-1 overexpression increased esterification into the glycerolipid pool at both concentrations of oleate in both groups of subjects . effect of pgc-1 overexpression on fatty acid incorporation in hskmc from lean and obese donors. n = 8) donors were incubated with either 100 mol / l (a, c, and e) or 500 mol / l (b, d, and f) oleate, and c - labeled incorporation into glycerolipid (a and b), tag (c and d), and dag (e and f) was determined. data are expressed as means se and significant differences denoted at the p 0.05 level. * significant difference between lean and obese for that treatment. significant main effect comparing control (-gal) and pgc-1 overexpression (ad - pgc-1) at the respective adenoviral doses. f. under control conditions, cells from obese subjects had increased oleate incorporation into tag (p < 0.05) (fig . 3e) in obese cells compared with lean cells when exposed to low oleate conditions. oleate incorporation into pl did not differ between lean and obese control cells regardless of oleate concentration (data not shown ; p > 0.05). compared with the control condition (-gal), pgc-1 overexpression increased oleate incorporation into tag (p < 0.05) (fig . 3c and d) and pl (data not shown ; p < 0.05) without affecting dag. pgc-1 overexpression did not normalize intramyocellular lipid incorporation into storage in hskmc from obese subjects to that of the lean individuals under the same treatment (fig . the rate of oleate incorporation into glycerolipids ( specifically, tag and dag) was higher when cells were exposed to the higher oleate concentration (p < 0.05) (fig . lipid partitioning was estimated by determining the rate of lipid esterification relative to complete fao ; a higher ratio is indicative of increased partitioning toward storage . myotubes from obese donors had a higher partitioning index compared with hskmc from lean donors ( p < 0.05) (fig . overexpression of pgc-1 at the low dose did not alter this ratio from the control condition, whereas high - dose pgc-1 overexpression ( approximately eightfold increase in pgc-1 protein) decreased this ratio under low oleate conditions (p < 0.05) (fig . pgc-1 overexpression does not normalize lipid partitioning rates between hskmc from lean and obese donors. the partitioning of fatty acids between oxidative and storage pathways was evaluated by dividing the rate of oleate esterified into glycerolipid by the rate completely oxidized in response to either 100 mol / l (a) or 500 mol / l (b) oleate in hskmc from lean (n = 8) and obese (n = 8) donors. data are means se and significant differences denoted at the p 0.05 level. * significant difference between lean and obese for that treatment. significant main effect comparing control (-gal) and pgc-1 overexpression (ad - pgc-1) at the respective adenoviral doses. mtdna, coxiv, and mttfa protein content were depressed (27, 35, and 43%, respectively ; p < 0.05) (fig . the overexpression of pgc-1 protein by 3.5-fold ( low dose) increased mtdna content by 27% in hskmc from lean and obese subjects, whereas increasing pgc-1 protein approximately eightfold increased mtdna content by 66 and 72% in myotubes from lean and obese individuals, respectively (p < 0.05) (fig . overexpression of pgc-1 increased coxiv and mttfa protein content in a dose - dependent manner ( p < 0.05) (fig . 5b and c). however, despite pgc-1 overexpression, mtdna, coxiv, and mttfa protein content still remained depressed in the cells from obese compared with lean donors under the same experimental treatment (p < 0.05) (fig . pgc-1 increases mtdna ( a), coxiv protein (b), and mttfa protein (c) in hskmc from lean and obese donors. mtdna, n = 9 for lean and obese; coxiv and mttfa, n = 8 for lean and obese. data are expressed as means se and significant differences denoted at the p 0.05 level. * significant difference between lean and obese for that treatment. significant main effect comparing control (-gal) and pgc-1 overexpression (ad - pgc-1) at the respective adenoviral doses. 6a and b ) or coxiv (fig . 6c and d) protein content, the decrement in fao evident with obesity (fig . pgc-1 overexpression had no effect on fao relative to mtdna ( p > 0.05) and decreased fao relative to coxiv protein content by 50% (p < 0.05) (fig . fao does not differ between hskmc from lean and obese donors when normalized to indexes of mitochondrial content. fao normalization to indexes of mitochondrial content were evaluated by dividing the rate of complete oleate oxidation from c - labeled incorporation into co2 (fao) by mtdna copy number per diploid nuclear genome (a and b) or by coxiv protein expression in arbitrary units (c and d) under both 100 umol / l (a and c) and 500 umol / l (b and d) oleate conditions. fao / mtdna, n = 9 for lean and obese; fao / coxiv, n = 8 for lean and obese. significant main effect comparing control (-gal) and pgc-1 overexpression (ad - pgc-1) at the respective adenoviral doses. findings from the dose - response experiment for ad - pgc-1 are presented in fig. 1. treatment of myotubes with ad--gal (control virus) had no effect on pgc-1 protein content compared with that on no - virus controls (fig . 1a). in hskmc derived from lean and obese individuals, pgc-1 protein (fig . 1a) and mrna (data not shown) increased in a dose - dependent manner with increasing pgc-1 adenovirus titer; there were no differences between pgc-1 protein content in hskmc from lean and obese individuals with or without overexpression. 1a, for subsequent experiments we selected the 1-ul dose (5 10 plaque - forming units / ml) as pgc-1 protein content was increased by a magnitude similar to that reported with endurance - oriented exercise training (3.5-fold increase in pgc-1 protein over control) and the 2-l dose (1 10 plaque - forming units / ml) to represent a supraphysiological increment (approximately eightfold increase in pgc-1 protein over controls) in protein content. protein levels of mttfa and coxiv, indicators of mitochondrial biogenesis, also increased in a dose - dependent manner with pgc-1 adenovirus (fig . ad - pgc-1 overexpression in cultured myotubes ( hskmc) from lean and obese donors. a: pgc-1 protein content in no - virus controls (nvc), ad--gal controls, and ad - pgc-1treated hskmc. b: mttfa and coxiv protein content increased dose dependently with increasing pgc-1 viral titer in hskmc. to evaluate the role of pgc-1 in regulating skeletal muscle lipid oxidation, we treated myotubes from lean and obese donors with either low or high ad - pgc-1 for 24 h, followed by 3 h incubation with either 100 or 500 mol / l oleate. in all experiments, fao from the no - virus controls did not differ from that in the ad--gal controls (data not shown). 2. under control conditions (low and high -gal), complete fao (co2 production) was consistently depressed by 30% in hskmc from obese individuals with either the 100 or 500 mol / l oleate incubation (fig . 2a, overexpression of pgc-1 by 3.5-fold ( low dose) ed in an 30% increase in complete fao (100 mol / l oleate) in hskmc derived from lean and obese donors. the eightfold increase in pgc-1 protein (high dose) also increased complete fao in cells from both lean (64%) and obese (70%) donors at the 100 mol / l oleate concentration (p < 0.05) (fig . although pgc-1 overexpression increased fao regardless of the category of the donor, absolute values for complete fao remained depressed in hskmc derived from obese compared with those in lean subjects under all conditions ( fig . similar findings were obtained in response to the higher oleate concentration ( 500 mol / l) with respect to differences between cell types and the effects of pgc-1 overexpression (fig . total fao, determined as the sum of incomplete ( asm) and complete oxidation did not differ between cell type under any conditions (fig . 2c and d), and pgc-1 overexpression increased total oxidation in a dose - dependent manner. total and complete fao were significantly higher at 500 mol / l oleate compared with 100 mol / l oleate in control and pgc-1treated cells (p < 0.05) (fig . d). effect of pgc-1 overexpression on fao and oxidation efficiency in hskmc from lean and obese donors. hskmc cultured from lean (n = 12) and obese (n = 9) donors was treated with either low- or high - dose recombinant ad--gal or pgc-1 and incubated with either 100 mol / l (a, c, and e) or 500 (b, d, and f) mol / l oleate. complete fao was measured from c - labeled incorporation into co2 (a and b). total fao (c and d) was measured as the sum of c - labeled incorporation into co2 and c - labeled incorporation into asms, with asm serving as an index of incomplete fao. oxidation efficiency was determined as the ratio of asm to complete fao, represented as asm / co2 (e and f), with higher values indicative of reduced efficiency. data are expressed as means se and significant differences denoted at the p 0.05 level. * significant difference between lean and obese for that treatment. significant main effect comparing control (-gal) and pgc-1 overexpression (ad - pgc-1) at the respective adenoviral doses. significant increase in lean subjects with pgc-1 overexpression compared with control at the respective adenoviral doses. a ratio of incomplete (asm) to complete (co2) oleate oxidation was calculated as an index of fao efficiency. in control cells treated with 100 mol / l oleate, radiolabel incorporation into asm relative to co2 was approximately twofold higher in myotubes from obese compared with lean individuals (p < 0.05) (fig . the high relative rate of incomplete oxidation in the obese compared with the lean group was retained regardless of the level of pgc-1 overexpression or oleate concentration ( fig . pgc-1 overexpression increased the asm - to - co2 ratio by 55% ( p < 0.05); pgc-1 overexpression in cells from obese subjects had no effect on this ratio (fig . the asm / co2 ratio was significantly higher at 500 mol / l oleate compared with 100 mol / l oleate in control and pgc-1treated cells ( p < 0.05) (fig . control cells ( -gal) from obese individuals had a greater rate of oleate incorporation into the glycerolipid pool in response to low (64%) and high (42%) oleate concentrations (fig . 3a and b). cells from obese individuals, regardless of treatment, consistently incorporated more lipids into the glycerolipid pool compared with hskmc from lean subjects (fig . 3a and b). pgc-1 overexpression increased esterification into the glycerolipid pool at both concentrations of oleate in both groups of subjects. effect of pgc-1 overexpression on fatty acid incorporation in hskmc from lean and obese donors. n = 8) donors were incubated with either 100 mol / l (a, c, and e) or 500 mol / l (b, d, and f) oleate, and c - labeled incorporation into glycerolipid (a and b), tag (c and d), and dag (e and f) was determined. data are expressed as means se and significant differences denoted at the p 0.05 level. * significant difference between lean and obese for that treatment. significant main effect comparing control (-gal) and pgc-1 overexpression (ad - pgc-1) at the respective adenoviral doses. f. under control conditions, cells from obese subjects had increased oleate incorporation into tag (p < 0.05) (fig . 3e) in obese cells compared with lean cells when exposed to low oleate conditions. oleate incorporation into pl did not differ between lean and obese control cells regardless of oleate concentration (data not shown ; p > 0.05). compared with the control condition (-gal), 3c and d ) and pl (data not shown ; p < 0.05) without affecting dag. this was evident in hskmc from both lean and obese subjects (fig . 3c pgc-1 overexpression did not normalize intramyocellular lipid incorporation into storage in hskmc from obese subjects to that of the lean individuals under the same treatment ( fig . 3c and e). the rate of oleate incorporation into glycerolipids (specifically, tag and dag) was higher when cells were exposed to the higher oleate concentration (p < 0.05) (fig . lipid partitioning was estimated by determining the rate of lipid esterification relative to complete fao ; a higher ratio is indicative of increased partitioning toward storage . myotubes from obese donors had a higher partitioning index compared with hskmc from lean donors ( p < 0.05) (fig . overexpression of pgc-1 at the low dose did not alter this ratio from the control condition, whereas high - dose pgc-1 overexpression ( approximately eightfold increase in pgc-1 protein) decreased this ratio under low oleate conditions (p < 0.05) (fig . pgc-1 overexpression does not normalize lipid partitioning rates between hskmc from lean and obese donors. the partitioning of fatty acids between oxidative and storage pathways was evaluated by dividing the rate of oleate esterified into glycerolipid by the rate completely oxidized in response to either 100 mol / l (a) or 500 mol / l (b) oleate in hskmc from lean (n = 8) and obese (n = 8) donors. data are means se and significant differences denoted at the p 0.05 level. * significant main effect comparing control (-gal) and pgc-1 overexpression (ad - pgc-1) at the respective adenoviral doses. 5. mtdna, coxiv, and mttfa protein content were depressed (27, 35, and 43%, respectively ; p < 0.05) (fig . the overexpression of pgc-1 protein by 3.5-fold ( low dose) increased mtdna content by 27% in hskmc from lean and obese subjects, whereas increasing pgc-1 protein approximately eightfold increased mtdna content by 66 and 72% in myotubes from lean and obese individuals, respectively (p < 0.05) (fig . overexpression of pgc-1 increased coxiv and mttfa protein content in a dose - dependent manner ( p < 0.05) (fig . 5b and c). however, despite pgc-1 overexpression, mtdna, coxiv, and mttfa protein content still remained depressed in the cells from obese compared with lean donors under the same experimental treatment (p < 0.05) (fig . pgc-1 increases mtdna ( a), coxiv protein (b), and mttfa protein (c) in hskmc from lean and obese donors. mtdna, n = 9 for lean and obese; coxiv and mttfa, n = 8 for lean and obese. data are expressed as means se and significant differences denoted at the p 0.05 level. * significant main effect comparing control (-gal) and pgc-1 overexpression (ad - pgc-1) at the respective adenoviral doses. as presented in fig. 6, when complete fao (co2 production) at either 100 or 500 mol / l oleate was expressed relative to mtdna (fig . 6c and d) protein content, the decrement in fao evident with obesity (fig . pgc-1 overexpression had no effect on fao relative to mtdna ( p > 0.05) and decreased fao relative to coxiv protein content by 50% (p < 0.05) (fig . fao does not differ between hskmc from lean and obese donors when normalized to indexes of mitochondrial content. fao normalization to indexes of mitochondrial content were evaluated by dividing the rate of complete oleate oxidation from c - labeled incorporation into co2 (fao) by mtdna copy number per diploid nuclear genome (a and b) or by coxiv protein expression in arbitrary units (c and d) under both 100 umol / l (a and c) and 500 umol / l (b and d) oleate conditions. fao / mtdna, n = 9 for lean and obese; fao / coxiv, n = 8 for lean and obese. significant main effect comparing control (-gal) and pgc-1 overexpression (ad - pgc-1) at the respective adenoviral doses. the intent of the current study was to determine whether the low rates of skeletal muscle fao observed in obese humans could be linked to a lower mitochondrial content in skeletal muscle and, given the role of pgc-1 in stimulating mitochondrial biogenesis, to examine whether increasing pgc-1 by a physiologically relevant increment could mitigate the foregoing deficits. hskmc was selected as the experimental model because myotubes established in culture from extremely obese donors display a reduction in fao that is quantitatively similar to that reported in skeletal muscle strips (60%), muscle homogenates (50%), and in vivo studies using c tracers (22%) or indirect calorimetry (40%). other reports have likewise demonstrated that interindividual variability in fat oxidation assessed in vivo in healthy young men was preserved in hskmc. 2 ) further establish the utility of hskmc as a model for studying fuel metabolism in human skeletal muscle. there is controversy whether the reduction in complete fao in human skeletal muscle observed with obesity can be attributed to the existing mitochondria being dysfunctional (i.e., altered morphology), the existing mitochondria being fully functional but expressed at a lower concentration with obesity, or a combination of both of these conditions. an important finding of the present study was that although complete fao and mitochondrial content were reduced in hskmc from extremely obese donors (figs . 2 and 5), when complete fao was normalized to indexes of mitochondrial content, the differences between lean and obese subjects were abolished (fig . the finding that the reduced complete fao in myocytes from obese donors is associated with reduced mitochondrial content ( fig . 6) suggests that the impairment in complete fao in human skeletal muscle with obesity may be attributed, at least in part, to a reduction in mitochondrial content (fig . this obesity phenotype is akin to characteristics exhibited by type 2 muscle fibers ; in support, a greater proportion of type 2 fibers has been reported with extreme obesity, although it is not evident whether all traits of glycolytic tissue (i.e., contractile properties) are retained in cell culture. while our data suggest that mitochondrial functionality remains intact with obesity, more extensive mitochondrial characterization (i.e., respiration data) is needed to provide definitive . in support of our findings, holloway et al. have reported that fao in mitochondria isolated from muscle biopsies was reduced with obesity when calculated on a whole - muscle basis; however, when normalized to mitochondrial protein content, fao was equivalent in lean and obese. the present findings add to the existing data in two novel ways. 2 ) and associated reduction in mitochondrial content (figs . 5 and 6) with obesity were evident even when mitochondrial content was manipulated via pgc-1 overexpression, indicating consistency. second, to our knowledge this is the first study to demonstrate a reduction in mitochondrial content in primary human muscle cell cultures from obese donors. it has been proposed that characteristics evident in hskmc have a genetic origin, given that any phenotype is retained as the cells proliferate and differentiate independently of in vivo influences. the current data thus provide the novel information that the reduction in mitochondrial content and the accompanying decrement in fao in skeletal muscle from extremely obese subjects consist of a heritable or imprinted component. master regulator coordinating mitochondrial biogenesis because elevating pgc-1 content activates critical downstream transcription factors, which effectively remodel the muscle cell to favor oxidative metabolism. accordingly, in l6 muscle cells the overexpression of pgc-1 enhanced fao and increased the expression of genes involved in oxidative processes; an elevation in pgc-1 is also believed to be a critical factor accounting for the increased fao and mitochondrial content in skeletal muscle with exercise training. in the present study, while pgc-1 overexpression increased fao and mitochondrial content independently of body composition, absolute values for complete fao and mitochondrial content remained depressed in the myocytes from obese subjects at both the physiological and supraphysiological pgc-1 doses (figs . 2 and 5). this novel indicates that skeletal muscle of obese individuals responds to signals triggered by pgc-1; however, the obese state appears to limit both mitochondrial biogenesis and oxidative capacity via mechanisms that are independent of pgc-1 abundance (figs . 2 and 5). the differences between the lean and obese myocytes in indexes of mitochondrial content (fig . 5) and fao (fig . 2) were relatively consistent with and without (control) pgc-1 overexpression; this suggests a possible reduction in another coactivator or mechanism involved with mitochondrial proliferation. mttfa protein content, a transcription factor downstream of pgc-1 critical for mtdna replication, mirrored the pattern of change seen in mitochondrial content (fig . 5), suggesting that the defect with obesity may involve this arm of pgc-1 coordination. a combination of variables such as pgc-1 cellular location (cytoplasm vs. nucleus), posttranslational modifications (i.e., acetylation and phosphorylation), and pgc-1 binding to or activation of transcription factors could contribute to the mitochondrial phenotype of obese hskmc. given the complexity of mitochondrial biogenesis, mechanisms independent of pgc-1 could also explain the obesity - associated decrement in fao and mitochondrial content. although the precise mechanism(s) are yet unknown, the present data indicate that extreme human obesity involves an intrinsic impairment in skeletal muscle mitochondrial biogenesis and content. we previously reported that exercise training increased complete fao and improved oxidation efficiency (co2 production / asm) to equivalent absolute values in lean and formerly extremely obese individuals despite an initial decrement in fao and elevated asm production in the obese subjects. 2, increasing pgc-1 content by both physiological and supraphysiological increments did not abolish the difference in complete fao and oxidation efficiency between lean and obese subjects as opposed to the total normalization seen with exercise training. this finding further suggests that other exercise factors, in addition to an increase in pgc-1 content, account for the improvement in skeletal muscle fao with physical activity in obese individuals. despite the reduced ability to completely oxidize lipid, obese cells exhibited similar rates of total oxidation, suggesting a downstream defect in the lipid oxidation pathway. suggested that an elevated asm / co2 ratio, as observed in cells derived from obese donors (fig . it is plausible that this occurred in the present study and that products of oxidative metabolism accumulated when downstream metabolic pathways could not adjust appropriately . in addition, obesity was linked to preferential partitioning of fa into the glycerolipid ( tag and dag) pools (fig . 3). these abnormalities are clinically relevant because both incomplete fao and intramuscular lipid accumulation have been implicated as markers and perhaps mediators of insulin resistance in obese individuals. interestingly, pgc-1 overexpression increased fatty acid incorporation into tag but not dag (fig . this finding is consistent with a recent report showing that transgenic mice with muscle - specific overexpression of pgc-1 have increased muscle tag when fed a high - fat diet. in this mouse model, pgc-1 overexpression ed in upregulation of diacylglycerol aclytransferase and mitochondrial glycerol-3-po4 acyl - transferase, two enzymes involved in tag synthesis; we speculate that similar mechanisms might be operative in the hskmc. thus did not rescue the cells from the obese subjects in terms of returning the indexes of lipid storage (figs . 3 and 4) to values seen in the cells from lean donors. in summary, skeletal muscle cells cultured from extremely obese donors exhibited depressed mitochondrial content, which could in turn be responsible for the diminished capacity to oxidize lipid and the preferential partitioning of lipid toward intramuscular storage with obesity. this phenotype may consist of a heritable or imprinted component because it is proposed that characteristics expressed in hskmc have a genetic origin. when pgc-1 was overexpressed in hskmc from lean and obese subjects, the mitochondrial phenotype of obesity persisted. this finding suggests a molecular impairment in mitochondrial proliferation that occurs independent or downstream of pgc-1 expression. additionally, pgc-1 overexpression did not fully recapitulate the effects of exercise training on fao in obese individuals, suggesting that additional mechanisms are involved with this intervention.

objectiveto determine whether the obesity - related decrement in fatty acid oxidation (fao) in primary human skeletal muscle cells (hskmc) is linked with lower mitochondrial content and whether this deficit could be corrected via overexpression of peroxisome proliferator activated receptor- coactivator-1 (pgc-1).research design and methodsfao was studied in hskmc from lean (bmi 22.4 0.9 kg / m2 ; n = 12) and extremely obese (45.3 1.4 kg / m2 ; n = 9) subjects. recombinant adenovirus was used to increase hskmc pgc-1 expression (3.5- and 8.0-fold), followed by assessment of mitochondrial content (mtdna and cytochrome c oxidase iv), complete (14co2 production from labeled oleate), and incomplete (acid soluble metabolites) fao, and glycerolipid synthesis.obesity was associated with a 30% decrease (p < 0.05) in complete fao, which was accompanied by higher relative rates of incomplete fao (asm production/14co2), increased partitioning of fatty acid toward storage, and lower (p < 0.05) mtdna (27%), coxiv (35%), and mitochondrial transcription factor (mttfa) (43%) protein levels. pgc-1 overexpression increased (p < 0.05) fao, mtdna, coxiv, mttfa, and fatty acid incorporation into triacylglycerol in both lean and obese groups. perturbations in fao, triacylglycerol synthesis, mtdna, coxiv, and mttfa in obese compared with lean hskmc persisted despite pgc-1 overexpression. when adjusted for mtdna and coxiv content, fao was equivalent between lean and obese groups.reduced mitochondrial content is related to impaired fao in hskmc derived from obese individuals. increasing pgc-1 protein levels did not correct the obesity - related absolute reduction in fao or mtdna content, implicating mechanisms other than pgc-1 abundance.

a 35-year - old male, diagnosed with obsessive - compulsive disorder (ocd) for the past 5 years was prescribed on fluoxetine up to 80 mg / day. he was receiving this treatment for the past 1 year with partial improvement of symptoms despite adequate compliance. cognitive behavior therapy was not possible due to transportation inconvenience. due to the persistence of symptoms, despite a high dose of fluoxetine, augmentation with clomipramine (up to 100 mg / day) after few days of the addition of clomipramine, the patient had reported dark pigmentation over face localized to both malar eminences. the dermatological consultation was sought for this hyperpigmentation; a diagnosis of melasma was made. his routine hemogram, thyroid function test, and serum cortisol levels were within normal limits. due to poor tolerance to clomipramine (excessive sedation and severe constipation), dose of clomipramine was reduced to 50 mg / day. due to the persistence of ocd symptoms, this ed in improvement in ocd symptoms over next 3 months; however, the skin pigmentation and severe constipation persisted. clomipramine dose was reduced to 25 mg / day for next 2 months and persistence of constipation ed in its stoppage. assessment score on naranjo adverse drug reaction (adr) probability scale was six, which was suggestive of probable adr. assessment on the world health organization - uppsala monitoring centre (who - umc) system for standardized case causality assessment also suggestive of probable adr with clomipramine. in our patient, the pigmentation is confined to the malar eminences of the face, with sparing of rest sun - exposed areas of the body. this adverse effect of clomipramine is not life - threatening, but can be a reason of concern from cosmetic sense. in our patient, clomipramine was not acceptable due to constipation and excessive sedation, although he was worried about melasma. stoppage of clomipramine ed in improvement of sedation, constipation as well as melasma. causality assessment is an important evaluation to establish the link between an adverse event with a particular drug. causality assessment has been done on naranjo adr probability scale and the who - umc system, which revealed probable adr (i.e., the adverse event followed exposure to drug and disappeared following drug discontinuation, it is unlikely attributable to disease or other drugs). in a study , it was found that the prevalence of severe adr being 1.4% in general; higher with the tricyclic antidepressant (tca) group and lower with monoamine oxidase inhibitors and selective serotonin reuptake inhibitors. among the tcas allergic exanthematous cutaneous reactions are known with tcas. however, there is description of a single case of 60-year - old female suffering from depressive episode, who developed pigmentation over light exposed areas of the body with the use of clomipramine. there are few case reports of such pigmentation with the use of imipramine, a congener of clomipramine. this is probably the second case study, reporting the rare side effect of clomipramine.

melasma is a hyperpigmented dermatological condition common in females. drugs such as steroids, cosmetics, and photosensitizing agents are known to cause melasma. we report here a case of an adult male with obsessive - compulsive disorder, receiving clomipramine, who developed melasma.

research is pivotal in the practice and development of medicine. it helps to discover new evidence, provides impetus to further research, draws attention to areas of interest and is instrumental in improving and innovating patient care. the obtained from research can be made available to the medical community either through publication in relevant journals or presentation at various forums. presentation at conferences can be either in poster or oral format, and aims for a rapid dissemination of the information to a large number of targeted professionals, stimulates discussion, encourages further research by young investigators, and is considered an important link between execution of a research protocol and publication of the completed work. presentation of research to a wide audience in conferences may lead to a change in clinical practice and opinion affecting the patient care, and hence the quality of research presentations should be maintained. a well - accepted marker of quality of the research that is presented is its success to be published subsequently in a peer - reviewed journal. the publication rate of presentations has been used to assess quality of research output in various fields of medicine including anesthesia. the publication rate varies from 11% to 78%, typically hovering around 30 - 50%. however, there is no data regarding the publication rate of research presented in anesthesia in an indian scenario. the indian society of anaesthesiologists hosts the national annual conference (isacon) that is the primary conference of anesthesia in india. the scientific committee of isacon selects papers to be presented at each annual conference after review of submitted abstracts. this observational study aimed to assess the publication rate of research presentations made in the isacon and also compare it with that of the annual meeting of american society of anesthesiologists (asa) held in the same year. as secondary observations, the site and timing of the full - text publication, completeness of the abstracts submitted to the conference, and changes between the abstract as accepted for conference proceedings and its full - text publication were also noted. the isacon publishes in print, abstracts of all poster and oral presentations made in the conference. the official website of asa allows free access to abstracts of all presentations. using these respective resources we identified abstracts of the presentations made at isacon and asa meetings. the mean time to publication after presentation has previously been noted to vary between 1.5 and 3 years, with a median of 18 months. thus, we conducted the present study using the proceedings of the conferences held in the year 2009. the total number of abstracts of research presentations made at isacon and asa meetings in this year was 363 and over 1700, respectively. the archive of abstracts of asa meeting allows access to an individual abstract one at a time, making manual sorting of such a large number of abstracts extremely difficult. to allow for comparison with the smaller number of presentations at the isacon we randomly selected an equal number of abstracts from the asa 2009 including all specialities, using a computer generated random number table and numbered abstracts. for calculation of the publication rate , we used a previous strategy to determine whether the presentation finally got published in a journal. herein, key words from the title and names of the first and last authors from the abstract were entered into pubmed as well as google scholar databases. key words were defined as the main elements in the title, and ranged from one to several in number. names of first and last authors were used, since they are usually the one who have contributed most to the paper and are likely to be the senior ones respectively, and thus named on any final publication. to standardize the of search process for full - text publication, only published papers that were similar with respect to hypothesis, study design, protocol, number of subjects and of the published abstract were considered as a match. if the published paper included the data presented in the abstract, along with additional data, it was still considered a match. at least two of the authors independently assessed the publications that were considered a match, to arrive at a consensus decision for accepting it as a full - text publication of the abstract. if the initial search did not reveal a full - text publication, all the key words from the title were entered before declaring an abstract unpublished. once matched as a full - text publication, the time of publication from its presentation was noted. it was also recorded whether the journal, wherein published, was indexed with pubmed. all abstracts, as published in the conference proceedings, were assessed for completeness by noting mention of aim, methods, quantitative , , study design (its specific mention), sample size (subjects recruited), and statistical tests. changes between abstract of the presentation and published paper with respect to the title, form (original research / case report / observational), authorship (number, order), number of study subjects, quantitative , and were also noted. the publication rate of abstracts from isacon and asa meetings was 19/363 (5%) and 80/363 (22%), respectively. percentage of full - text publications published in journals indexed with pubmed was 58% and 100% for the isacon and asa meetings, respectively. of the 363 abstracts presented at isacon while 270 (74.4%) were original research, 81 (22.3%) were case reports, and 12 (3.3%) were personal experiences or narratives. compared to this, there were no case reports among the asa meeting abstracts. the median (range) time to publication as full - text articles was 12 months and 14 months for isacon and asa meetings, respectively. from among the published articles, 26% from isacon and 0.07% of asa there was a difference in completeness of the abstracts accepted for presentation in the conferences. abstracts complete with respect to inclusion of aim, statistical tests, quantitative and were significantly higher in the asa meeting as compared with the isacon (p < 0.05 ; table 1). in contrast, the percentage of abstracts mentioning study design were greater from isacon meeting (p < 0.05); and those with sample size and methods were similar between the two conferences. completeness of abstracts of presentations a similar percentage of the final full - text publications from isacon and asa meetings (84% and 85%, respectively) differed from the initial abstract. with regard to the individual components, significantly higher percentage of publications from asa meeting had a change in the form, order of authors, , and (p < 0.05, table 2). the commonest change in the final manuscripts ing from both conferences was a change in the authorship. the increase in number of authors ranged from 1 to 9 and 1 to 8 in manuscripts originating from presentations of isacon (68%) and asa meetings (73%), respectively. we searched both pubmed and google scholar for locating full - text publications of the presented abstracts so as to include both indexed as well as nonindexed journals. previously, a publication rate of 31 - 43.6% has been noted from presentations made in international conferences in anesthesia. the publication rates noted in our study for both, the indian isacon (5%) and international asa meeting (22%) are lower than the previous figures. for suitable comparison of the rate for isacon , there is no previous data regarding publication rate from an indian conference in anesthesia. however, publication rate from indian conferences held in other medical specialities note higher publication rates than isacon; 16.5% in ophthalmology. the publication rate of asa meeting is only for the randomly selected sample of abstracts. low publication rates from authors failing to write the full - text manuscript or its being rejected by a journal during peer review. failure to write the manuscript could be related to several reasons including time constraints, the extra work required to achieve a full text publication, or a simple lack of motivation to achieve publication. also, in a usual indian scenario, while a presentation may be required to attend and obtain reimbursement of expenses for the conference, a publication is not required for the same. several times the work is presented by students / residents who have changing interests or insufficient time, preventing production of complete manuscript. the inclusion of a large number of case presentations in isacon (22.3%) may also contribute to the lower publication rate. to publish case reports is currently difficult, with most international journals having uniformly stepped down the space dedicated to case reports. this is because case reports are now relegated to the lowest class of evidence - based research and also affect the journal s impact factor adversely. although there are certain journals devoted to publishing case reports such as the journal of case reports, these are internet - based, open - access journals demanding a high publishing fees. stringent conditions on presentation of case reports in isacon need to be applied, and only a limited percentage of total presentations should be allocated to them. case reports that can convey novel management techniques or descriptions need to be considered. those with a beneficial outcome by applying previously described approaches in uncommon diseases should be specially desisted from. other suggestions to increase publication rate from isacon include introduction of formal training in research methodology and bioethics, as well as scientific paper writing during graduate and post - graduate courses. faculty members can be allocated structured time for research distinct from academic, clinical, and administrative responsibilities. better infrastructure or support for research such as free access to journals and secretarial assistance should also be made available. some of these are affirmative steps that can even be taken at departmental level without necessitating institutional policy decisions, making them feasible and sustainable. to augment the publication of research, ultimately, the will and efforts are to originate from all concerned including the authors. from among the published articles, 26% from isacon and 0.07% of asa these could be presentations that were submitted for consideration for publication at the time of the conference, or those already published, the latter being highly undesirable. a significantly lower percentage of abstracts from isacon were complete with regard to aim, statistical tests, quantitative , and as compared with asa meeting. besides this quantitatively higher percentage of isacon abstracts being incomplete, there are certain qualitative differences. there were a high number of abstracts from isacon that lacked quantitative , i.e., 177 of the 270 original research as compared with asa meeting (2/363 original research). this may be because these studies may not have been completed by the time of submission for presentation. the asa website includes the precondition of providing quantitative and and puts a word limit for each specified section, whereas the guidelines in the isacon website are less rigorous. it is also worrisome that two abstracts from isacon were accepted despite lack of aims or methods. these findings suggest the need for a more rigorous peer review of the abstracts submitted to isacon for potential presentation. a standardized protocol for screening and selection of abstracts needs to be put in place, that may help reduce any subjective bias on part of the reviewers. like certain other conferences, submission of a nonrefundable nominal fee may also help improve the quality of research being submitted to isacon. a similar percentage of full - text publications from both conferences had changes made in comparison to components of the original abstract. the commonest change between full - text publication and its abstract from conference proceedings was in the authorship, with an increase in authors ranging from 1 to 9 in isacon and 1 to 8 in asa meeting publications. change in authorship between presentation and publication may be due to the inclusion of an additional author if there is further analysis of data involving another investigator, or removal of an author if their involvement in manuscript preparation is below standards set for authorship by peer - reviewed journals. gift authorship that is prevalent may also explain the change in number or order of authorship. the limitations of our study include the possibility of some articles still pending for publication, missing articles published in journals not either in google scholar or pubmed, and typographical errors in the proceedings of isacon or asa meeting, which may have lead to the article being unavailable on search. also, since the computerized search for full - text articles was carried out manually the chances of human error can not be ruled out. to conclude, the publication rate from isacon (5%) is lower than that from asa meeting (22%). abstracts submitted to isacon lack completeness with respect to central aspects of research such as mentioning of methods, statistical methods, quantitative , and ; necessitating more rigourous guidelines and review prior to their acceptance. augmenting this quality check will also help to increase the publication rate eventually. lastly, changes are common in published full - text manuscripts as compared with their presented abstracts, implying that the presentation of research at conferences such as isacon helps in its improvement and augments publication.

: to assess the quality of research presentations made in conferences, its success or failure to be published in a peer - reviewed journal is a well - accepted marker. however, there is no data regarding the publication of research presentations made in indian conferences of anesthesiology.objective:the primary objective was to determine publication rate of research presented at the largest and best attended national conference in anesthesiology, the indian society of anaesthesiologists conference (isacon), and also compare it with the rate from an international conference american society of anesthesiologists (asa annual meeting) held in the same year.materials and methods: all 363 abstracts presented as poster or podium presentations at the isacon, and an equal number of randomly selected abstracts presented at asa annual meeting were searched on pubmed and google scholar for their full - text publications in peer - reviewed journals using a standardized search strategy. as secondary observations, abstracts were assessed for completeness by noting certain components central to research methodology. also, changes between abstract of the presentation and published paper were noted with respect to certain components.:the publication rate of presentations at isacon and asa meetings was 5% and 22%, respectively. the abstracts from isacon lacked central components of research such as methods and statistical tests. the commonest change in the full - text publications as compared with the original abstract from both conferences was a change in authorship.:steps are required to augment full - text publication of indian research, including a more rigorous peer review of abstracts submitted to isacon to ensure their completeness.

in the previous issue of critical care nathani and colleagues have assessed kl-6, a specific marker of type 2 alveolar epithelial cell injury, as a biomarker in acute lung injury (ali). biomarkers allow identification of patients at risk of developing disease or can be used as surrogate measures for clinical outcomes. additionally, measuring biological markers may be a valuable tool in understanding disease pathogenesis. in ali, the alveolar capillary barrier is disrupted and the alveolar epithelial cell function is critical to the recovery from ali / acute respiratory distress syndrome (ards). this knowledge provides a rationale for measurement of alveolar epithelial cell injury using surrogate biochemical measures such as kl-6, as a biomarker of ali. in the study of nathani and colleagues, plasma and bronchoalveolar lavage samples were collected following inclusion and on day 4 from 30 ventilated ali patients, from 12 patients at risk of developing ali and from 10 nonsmoking volunteers free of respiratory disease the study therefore had the benefit of allowing the investigators to look at kl-6 both in physiological and pathological states. the important findings from the study are that plasma kl-6 levels are increased in patients with ali, plasma kl-6 correlates with the severity of lung injury and plasma kl-6 is significantly elevated in nonsurvivors compared with survivors. furthermore bronchoalveolar lavage kl-6 is elevated in patients with ali and is higher in nonsurvivors. these findings extend the previous data showing kl-6 is elevated in plasma and epithelial lining fluid in ali. in relation to kl-6 in ali, questions that still remain to be answered include the specificity of the type 2 epithelial cell as the source of kl-6 as well as a need to confirm whether kl-6 elevation reflects epithelial cell injury, regeneration or secretion in response to inflammatory mediators. in addition, mechanical ventilation is known to cause epithelial injury, and an important area in which biomarkers may be valuable is in the assessment of ventilator - associated lung injury. increased surfactant protein d is associated with injurious ventilation strategies, and it would be interesting to know the effects of mechanical ventilation on kl-6. regardless of these outstanding questions, nathani and colleagues' work together with other data showing that elevated surfactant protein d, a type 2 epithelial cell product, is associated with a worse clinical outcome in ali / ards and that the receptor for advanced glycation end - products, an alveolar type 1 epithelial cell - associated protein, is increased in patients with ali implicates epithelial cell damage as an important determinant of outcome and implies the potential for alveolar epithelial cell biomarkers to predict outcome in ali. further, these data support the central role of epithelial injury in the pathogenesis of ali. notable limitations of all these surrogate biomarkers exist; they do not directly measure epithelial function, and they require laboratory analysis and therefore can not be performed by a clinician at the bedside. additionally, there is no biomarker of epithelial function that reliably identifies patients at risk of ali who will develop ali. the resolution of pulmonary oedema from the alveolar space, which is dependent on alveolar fluid clearance, is critical to the recovery from ali / ards. although this can not be measured at the alveolus, the potential difference can be measured readily across the nasal epithelium. measuring nasal potential difference is a simple noninvasive measurement easily undertaken repeatedly at the bedside. in an animal model, measurement of nasal potential difference correlated with alveolar fluid clearance. this observation supports the hypothesis that nasal potential difference measurement may be a surrogate marker for alveolar epithelial function. furthermore, premature infants with pulmonary oedema and patients susceptible to high - altitude pulmonary oedema have reduced nasal transepithelial sodium resorption, as measured by the baseline nasal potential difference, indicating that nasal potential difference measurement may be able to identify patients at risk of developing ali. although further work validating such functional measures of epithelial activity is required, it is probable that as well as biochemical measures such as kl-6, as demonstrated by nathani and colleagues, future biomarkers in ali will combine both biochemical and functional measures.

studies have indicated that measuring biochemical measures of epithelial injury in plasma and alveolar fluid may be useful in predicting outcome in acute lung injury. the present commentary briefly reviews the evidence supporting the use of these biochemical biomarkers of epithelial injury in acute lung injury, and in particular kl-6, as well as their limitations. the article additionally proposes the need for physiological markers of epithelial function to complement current biochemical biomarkers.

autoimmune pancreatitis (aip) is a distinct type of pancreatitis with a presumed autoimmune etiology. its clinical features, treatment, and prognosis are significantly different from those of non - aip chronic pancreatitis (cp). presently, epidemiological information about aip mainly comes from japan, south korea, the united states, and other countries. the morbidity of aip is 2.2 per 100 000 population in japan. in north america, among cp cases, , it is estimated that there are thousands of patients with aip in china, but only few cases are reported because most aip patients in china have been misdiagnosed and consequently not correctly treated. in a report of 36 aip patients in china, 18 (50%) cases were misdiagnosed as pancreatic cancer and 10 (28%) as non - aip pancreatitis. thus, further studies are needed to analyze the characteristics of aip in china and distinguish it from non - aip cp. the participation of cd4+cd25 + regulatory t cells (tregs) in the pathogenesis of the igg4 reaction in aip has been proposed. it was suggested that tregs might be involved in aip through in situ production of interleukin-10 (il-10) and transformation growth factor (tgf-), which could be followed by igg4 class switching and fibroplasia. therefore, forkhead box p3 (foxp3), as a good marker of cd4+cd25 + tregs, was analyzed to investigate the significance of cd4+cd25 + tregs in type 1 aip. it has been reported that il-17 plays a key role in the fibrosis of chronic inflammation. increasing il-17 expression was also reported as being involved in the pathogenesis of igg4-related sclerosing sialadenitis. type 1 aip is an igg4-related systematic autoimmune disease with dense fibrosis in the pancreas, but il-17 expression remains unclear in type 1 aip. in this study , we analyzed the clinical features of type 1 aip, detected the immunohistochemical expressions of foxp3 and il-17 in type 1 aip, and compared them with non - aip cp to improve the understanding of aip and identify factors for differentiation of the 2 diseases. because diagnosis of aip is primarily based on pathological features, clinically suspected type 1 aip and non - aip cp cases with pancreatic specimens were all reviewed at sun yat - sen memorial hospital from january 2000 to december 2013. the diagnosis of type 1 aip was according to icdc. dhall et al. reported that using a cutoff of 50 igg4-positive cells / hpf, the sensitivity and specificity for type 1 aip vs. other types of pancreatitis was 84% and 100%. another recent study found high levels of igg4 staining (> 10 igg4 + cells / hpf) in 17 of 20 (85%) aip pancreatic and extrapancreatic specimens compared with 1 of 175 (0.6%) control specimens, and positive igg4 staining enabled a definitive diagnosis in 91% of aip patients. in our study, 37.9% cases had > 50 igg4 + cells / hpf and all the type 1 aip patients had > 10 igg4 + cells / hpf. by contrast, in non - aip cp group, 45% cases had no igg+ cell infiltration, and others had < 10 igg+ cells / hpf. , showing that numerous igg4 + cell infiltration was helpful to differentiate the diagnosis of type 1 aip and non - aip cp. foxp3 is a specific marker of cd4+cd25 + tregs, which play a critical role in immune tolerance.. a higher expression level of foxp3 mrna in tissue was observed in igg4-related sclerosing pancreatitis and cholangitis, as well as larger infiltrates of cd4+cd25 + treg cells at involved organs and increased numbers of cd4+cd25 treg cells in the blood. furthermore, tregs can produce il-10 and tgf-, which could be followed by igg4 class switching and fibroplasia. our showed that foxp3 + cells were significantly more frequent in type 1 aip compared with non - aip cp. a positive correlation of foxp3 + with igg4 + cells was also observed. these suggest that foxp3 + cells might be involved in the pathogenesis of type 1 aip and can be helpful in distinguishing type 1 aip from non - aip cp. il-17 is a proinflammatory cytokine mainly produced by th17 cells, which plays an important role in the fibrosis associated with chronic inflammation, multiple sclerosis, and rheumatoid arthritis. il-17 can induce the production of many cytokine (such as il-16, tgf-, and granulocyte colony - stimulating factor), attract neutrophils, and cause massive inflammation and fibrosis. increasing il-17 expression however, our study indicated that il-17 + cells in type 1 aip were similar to those in non - aip cp, perhaps because both type 1 aip and non - aip cp have marked fibrosis and il-17 is associated with the fibrosis of chronic inflammation. moreover, il-17 + cells had no correlation with igg4 + cells. these indicate that il-17 + cells might not be useful for differentiation between type 1 aip and non - aip cp. furthermore, only cases with pathological specimens are included, which ensured the correct diagnosis, but could have excluded type 1 aip in cases without pathological specimens and cause selection bias. therefore, further prospective studies with larger sample size are needed to confirm the of this study. type 1 aip is a unique type of chronic pancreatitis and has distinctive symptoms, serological, imaging, and pathological characteristics, which could be used for differentiation from non - aip cp. foxp3 + cells might be helpful to distinguish type 1 aip from non - aip cp.

autoimmune pancreatitis (aip) is a distinct type of pancreatitis associated with a presumed autoimmune mechanism. the aim of this study was to analyze the clinical features and expressions of forkhead box p3 (foxp3) and interleukin-17 (il-17) in type 1 aip in china and to identify factors for differentiation of aip from non - aip chronic pancreatitis (cp).material / methodswe retrospectively reviewed pancreatic specimens with diagnosis of type 1 aip and non - aip cp at sun yat - sen memorial hospital in china from january 2000 to december 2013. the clinical symptoms, serological data, imaging findings, histopathology, and immunohistochemical findings of foxp3 and il-17 in the 2 groups were analyzed.twenty-nine patients with type 1 aip and 20 patients with non - aip cp were enrolled. obstructive jaundice was more common in type 1 aip than in non - aip cp (62.1% vs. 30.0%, p=0.042). the diffuse or segmental enlargement of the pancreas was more frequent in type 1 aip than in non - aip cp (72.4% vs. 40.0%, p=0.038). histopathology of type 1 aip presented dense lymphoplasmacytic infiltration, snowstorm - like fibrosis and abundant immunoglobulin (ig) g4 + cells. foxp3 + cells were more frequently observed in type 1 aip than in non - aip cp. il-17 + cell infiltration was similar between the 2 groups. furthermore, a positive correlation was found between foxp3 + and igg4 + cell counts in the pancreas of patients with type 1 aip.type 1 aip has distinctive symptoms, image, and pathological characteristics, which could be used for differentiation from non - aip cp. foxp3 + cells might be helpful to distinguish type 1 aip from non - aip cp.

the relatively small and non - repetitive nature of microbial genomes, coupled with the rapid advancement of sequencing technology in the last decade, have led to the generation of a staggering amount of bacterial genome records. the national center for biotechnology information (ncbi) genome database currently maintains genome records for over 3000 high quality reference and representative genome assemblies and another 50 000 incomplete assemblies. the existing collections of genomes are just the beginning; the earth microbiome project http://www.earthmicrobiome.org ) is in the early stages of analyzing and cataloguing over 200 000 environmental samples from around the world, and estimates that this will in the sequencing of 500 000 reconstructed microbial genomes. making sense out of this abundance of data, while a daunting challenge, will generate a wealth of knowledge for the microbial and human genomic research community. for microbial genomes, as well as most other biological data, knowledge is distributed across resources that occupy the full spectrum from very large, broad coverage, centralized, major government - funded institutions such as ncbi (www.ncbi.nlm.nih.gov/gene, ) and uniprot (www.uniprot.org, ) to boutique, topic - focused databases like the odb3 database of prokaryotic operons (www.http://operondb.jp/, ), to the unstructured primary literature. the ability to process data smoothly from across that spectrum would greatly increase the efficiency of microbial research giving the researcher the ability to explore questions, based on existing evidence - based data, before any experimentation. what other microorganisms influence the persistence of an infection by a human pathogen such as chlamydia, and by what mechanism?. an expert may generate hypothetical answers to this question by blending their knowledge with information spread through the literature and various databases. as an example, the statements illustrated in figure 1, originating from multiple sources, including primary literature, and structured databases such as ncbi gene, uniprot, drugbank (www.drugbank.ca/, ) and brenda (http://www.brenda-enzymes.info/, ), link together to yield the hypothesis that co - infection by prevotella spp. , clostridiales spp. and escherichia coli in the vaginal microbiome increase the persistence of infection through the generation of indole, a key substrate in the tryptophan biosynthesis pathway. experts, like caldwell and colleagues, have done the experiments and the literature research to generate the hypothesis that there is a greater risk of clearance failure, leading to persistent infection that should be treated appropriately, when these other indole - creating microbes are present. figure 1.illustration of the complex network of interacting entities between human, chlamydial, and other microbial species in the urogenital microbiome. when a human epithelial cell is infected by c. trachomatis, it responds by depleting the cell of l - tryptophan, an essential amino acid for chlamydial growth, through ifn- mediated expression of the tryptophan degrading enzyme indoleamine 2,3-dioxygenase (ido)(orange). ido degrades tryptophan to n - formylkynurenine, a tryptophan precursor that c. trachomatis is not capable of converting into tryptophan. often this clears the infection, but episodically c. trachomatis rescues itself from this host defense by converting exogenous indole into l - tryptophan through gene expression regulated by its trp operon. several experiments support the hypothesis that the likely source of exogenous indole is from other microbes in a perturbed vaginal microbiome; as part of l - tryptophan degradation via the pyruvate pathway. microbes producing tryptophanase (yellow), an enzyme that degrades l - tryptophan to indole and pyruvate are commonly found in the urinary tract of patients also presenting with bacterial vaginosis (bv). examples of indole producers, commonly associated with bv in the female urogenital tract include prevotella spp., e. coli and clostridiales spp. blue urls indicate the various resources that maintain the data. the arrows between entities indicate the properties used to define their relationships once aggregated in wikidata. illustration of the complex network of interacting entities between human, chlamydial, and other microbial species in the urogenital microbiome. when a human epithelial cell is infected by c. trachomatis, it responds by depleting the cell of l - tryptophan, an essential amino acid for chlamydial growth, through ifn- mediated expression of the tryptophan degrading enzyme indoleamine 2,3-dioxygenase (ido)(orange). ido degrades tryptophan to n - formylkynurenine, a tryptophan precursor that c. trachomatis is not capable of converting into tryptophan. often this clears the infection, but episodically c. trachomatis rescues itself from this host defense by converting exogenous indole into l - tryptophan through gene expression regulated by its trp operon. several experiments support the hypothesis that the likely source of exogenous indole is from other microbes in a perturbed vaginal microbiome; as part of l - tryptophan degradation via the pyruvate pathway. microbes producing tryptophanase (yellow), an enzyme that degrades l - tryptophan to indole and pyruvate are commonly found in the urinary tract of patients also presenting with bacterial vaginosis (bv). examples of indole producers, commonly associated with bv in the female urogenital tract include prevotella spp., e. coli and clostridiales spp. blue urls indicate the various resources that maintain the data. the arrows between entities indicate the properties used to define their relationships once aggregated in wikidata. by pulling these pieces of knowledge together into a common database, with defined connections between them, a list of the taxa involved can be generated as candidate answers to the above question with a single query. once this is achieved and new data is added, the network grows and the collective benefit grows as well. the complicated and disordered is given order in a central container with a mechanism for sifting through it, giving the microbial researcher a powerful tool for making sense of the published data. model organism databases such as the mouse genome database (http://nar.oxfordjournals.org/content/43/d1/d726.short) mouse genome informatis (mgi) (http://www.informatics.jax.org), would greatly aid researcher s ability to unlock connections between microbes and the organisms they interact with. however, such large data warehouses are typically maintained by expensive teams of data and domain experts. the immense scale of microbial data is economically incompatible with this kind of centrally funded approach, and the same resolution would not be achieved. in one way or another, the greater scientific community, encompassing both active scientists and interested members of the general public must be empowered to contribute their mental energy in a community - wide collaborative effort. here wikidata is a new, centralized, yet openly editable platform for semantic knowledge representation that is maintained by the wikimedia foundation (the same entity that maintains all of the 200 + different language wikipedias). centralizing structured knowledge in this open database generates the opportunity to distribute the labor of data curation across a far broader community than was before realistic. in doing so , it offers a new approach to the knowledge integration problem that is ideally suited to the challenge of representing the exploding body of information about microbial genomics. here, we describe the initial work of building a wikidata - based representation of microbial genetics. a centralized resource for microbial genomics will need to capture a wide variety of different kinds of entities and relationships to support useful queries. rather than attempt to build a system that models all of this complexity up - front , we are taking the approach of seeding the openly extensible wikidata database with the beginnings of this model and thus encouraging the broader community to see the opportunity to collaborate on its evolution. its schema - free nature naturally supports data model changes and its open, wiki - based nature supports constructs such as watchlists and wikiprojects that have proven effective in facilitating the attainment of community consensus over time in other open projects such as the genewiki. on a technical level it has no overarching schema to ordain what kind of data elements can be linked together. in contrast to a standard relational model, this empowers database contributors to freely expand connections as they see fit. the only constraints on the structure of the graph are socially imposed. to add new content to wikidata, would - be editors need to either use existing properties or make a request to the community that new properties be created. property requests are openly discussed for a period of time until a consensus has been reached on their value to the system and how they should be applied. this request process allows the community to control the emerging structure of the graph and reduce problems that might arise from completely unrestricted editing. as a starting point for seeding the collaborative creation of a centralized microbial database in wikidata, we established the structures needed to represent the entities and relations depicted in figure 1. in the context of wikidata, this work amounts to the creation of a set of items and properties that are used to describe features of those items. a wikidata item is defined by a unique identifier (e.g. q131065), a label (i.e. chlamydia trachomatis), a description (i.e. species of prokaryote) and a set of claims about the item organized into statements (figure 2). a statement consists of a triple with an item as the subject, a wikidata - defined property as the predicate (i.e. taxon rank, property p105), and another wikidata item or literal data value as the object. optionally, a set of references can be added as evidence and provenance for the claim made by the triple, and qualifiers can specify the context where the claim is valid (https://www.mediawiki.org/wiki/wikibase/data mode l / primer). figure 2.example wikidata item. a wikidata item defined by its label, description and statements that provide annotations and create relationships with other items in the database. example wikidata item. a wikidata item defined by its label, description and statements that provide annotations and create relationships with other items in the database. the ontology of wikidata is determined by the set of properties that may be used to create claims about the items within it. entities can be created at any time, but properties can only be created by elected administrators following community discussion. the properties needed to support our current data model are listed in table 1. it is worth noting that most of these properties are either generic (i.e. subclass of) or were defined by the molecular biology wikiproject (https://www.wikidata.org/wiki/wikidata:wikiproject_molecular_biology) before the outset of the present work on microbial genomes. likewise , many requisite items, e.g. entities representing drugs and enzymes, were also present at the outset of the project and continue to be improved by members of the community. already, wikidata is showing how an open system can evolve over time with subsequent efforts building directly on prior work. table 1wikidate propertiesidnamevalue typep685ncbi taxonomy idstringp105taxon rankwikidata itemp171parent taxonwikidata itemp2249refseq genome idstringp1542cause ofwikidata itemp351entrez gene idstringp279subclass ofwikidata itemp703found in taxonwikidata itemp644genomic startstringp645genomic endstringp702uniprot idstringp637refseq protein idstringp702encoded bywikidata itemp688encodeswikidata itemp680molecular functionwikidata itemp681cellular componentwikidata itemp682biological processwikidata itemp361part ofwikidata itemp128regulateswikidata itemp1056productwikidata itemp2175medical condition treatedwikidata itemp2176drug used for treatmentwikidata item some of the general purpose properties such as product, part of, cause of and regulates are currently used to establish the connections in figure 1, but are likely to be replaced or extended with more biology - specific relations (such as precursor and substrate of) over time. the other aspects of the current model that are more specific to representing microbial data are depicted in figure 3. the basic framework of the microbial genetic data model in wikidata showing items and the statements that connect them. item types are demarcated by label and color (i.e. gene item = blue and protein item = orange). data model template. the basic framework of the microbial genetic data model in wikidata showing items and the statements that connect them. item types are demarcated by label and color (i.e. gene item = blue and protein item = orange). one key requirement for modeling microbial data microbiome and genomic research require the ability to do both intra- and interspecies comparative analysis. to support this work , our model follows a hierarchical taxonomy ranking scheme with the microbial species assigned to a wikidata item (i.e. c. trachomatis # q131065) defined by the core properties ncbi taxonomy id (p685), since genome annotations are based on the genome assembly of the specific strain sequenced and that genome assembly has its own unique identifier (i.e. ncbi refseq genome accession number), strain level distinction is critical in bacteria. q20800373 ) include the same core properties as a species item, the refseq genome id (p2249), and are linked to the species item via the parent taxon , the gene and protein must be kept as distinct entities, while maintaining their connections for queries down the line. a microbial gene item contains the similar core properties of a human gene item, including entrez gene id (p351) and subclass of (p279), but, found in taxon (p703) was added to distinguish which strain / genome assembly this particular gene came from. the gene links to its product item via the encodes (p688) property and reciprocally, the protein item will link to the gene that encoded it by the encoded by (p702) property. core properties for microbial protein include refseq protein i d (p637), uniprot id (p352), found in taxon (p703) and subclass of (p279). functional annotations are downloaded from the uniprot protein record and included here as subclasses of the gene ontology terms, molecular function (p680), cellular component (p681) and biological process (p682). given the data model depicted in figure 3 and encapsulated in the properties listed in table 1, we have seeded wikidata with representative content for 21 species of bacteria totaling 43 694 gene and 37 966 protein items, from various public databases. bot, a program for making automated edits in wikidata, with source code available at www.bitbucket.org/sulab/wikidatabots/src. in addition, we manually established all of the wikidata items and relationships needed to realize the operon data structure in figure 1. this information can be accessed through the various apis offered by wikidata (https://www.wikidata.org/w/api.php, https://query.wikidata.org/). as an example , a user can easily retrieve all genes, proteins, and gene ontology annotations for the two strains of chlamydia that are currently loaded using a wikidata sparql query (figure 4). figure 4.sparql query for all microbial genes, proteins and associated gene ontology annotations in wikidata. properties used: p351 = entrez_gene_id, p688 = encodes, p703 = found in taxon, p352 = uniprot_id, p171 = parent taxon. note that the * operator on p171 * in a recursive search for organisms that descend from wd: q10876 (bacteria). this query may be executed at https://query.wikidata.org/. sparql query for all microbial genes, proteins and associated gene ontology annotations in wikidata. properties used: p351 = entrez_gene_id, p688 = encodes, p703 = found in taxon, p352 = uniprot_id, p171 = parent taxon. note that the * operator on p171 * in a recursive search for organisms that descend from wd: q10876 (bacteria). this query may be executed at https://query.wikidata.org/. note that the query actually requests this information for all bacteria through the? taxa wdt: p171 * (parent taxons) wd: q10876 (bacteria) aspect of the query. as more bacterial genomes are loaded by us or other groups, the same query will return more and more data. as another example, the following sparql query returns all operons, their regulators and their products (figure 5). figure 5.sparql query for all operons, their regulators, the taxon that expresses them and their functional products in wikidata. q139677 is the wikdata item for the class operon. properties used: p279 = subclass of, p527 = has part, p1056 = product, p128 = regulates, p688 = encodes, p703 = found in taxon. this query may be executed at https://query.wikidata.org/. sparql query for all operons, their regulators, the taxon that expresses them and their functional products in wikidata. q139677 is the wikdata item for the class operon. properties used: p279 = subclass of, p527 = has part, p1056 = product, p128 = regulates, p688 = encodes, p703 = found in taxon. this query may be executed at https://query.wikidata.org/. revisiting the example question illustrated in figure 1 regarding organisms that are likely to be related to the persistence of chlamydial infections, we can ask what microbes are located in the female urogential tract and capable of generating indole (figure 6). this query returns e. coli iai39, a bacterial strain commonly found in patients with a perturbed vaginal microbiome. this query implicates an additional microbe suspect in chlamydial evasion of the host immune response. with disease drug mappings in wikidata, imported from the national drug file - reference terminology (ndf - rt) database, queries that identify antibiotic compounds with action against e. coli, c. trachomatis or both are possible (figure 7). these queries could inform an alternative treatment strategy that also targets the indole - producing bacteria in the vaginal microbiome, as was suggested by the original authors of the hypothesis. figure 6.sparql query for all organisms that are located (p276) in the female urogential tract (wd : q5880) and that have a gene with product (p1056) indole (wd : q319541). this query may be executed at https://query.wikidata.org/. figure 7.sparql query for all drugs used for treatment (p2176) for commensal e. coli infectious disease (q18975220), chlamydia infection (q153356) and both e. coli and chlamydia infections with a venn diagram display of the . these queries may be executed at https://query.wikidata.org/. sparql query for all organisms that are located (p276) in the female urogential tract (wd : q5880) and that have a gene with product (p1056) indole (wd : q319541). this query may be executed at https://query.wikidata.org/. sparql query for all drugs used for treatment (p2176) for commensal e. coli infectious disease (q18975220), chlamydia infection (q153356) and both e. coli and chlamydia infections with a venn diagram display of the . wikidata is certainly not a replacement for core data curation centers such as ncbi and uniprot. but it could form the basis for a complementary, stable and cost - effective approach for capturing content that is either left trapped in the literature or represented only in small databases subject to the perils of funding cuts and general link rot. though the microbial queries listed above currently return only a small fraction of the relevant content that exists in the world , the power of the wikidata approach is that our seedling database can be extended by anyone with the will to do so. wikidata is now edited by > 15 000 active users and currently has over 15 million content pages (https://www.wikidata.org/wiki/special:statistics). because of its open structure, its change tracking features and its evidence - capturing data model, it encourages community participation at all levels. while the community consensus building process can be slow and at times frustrating, it drives the stability and quality of wikidata content. even government - backed institutions like ncbi and european bioinformatics institute (ebi) (http://www.ebi.ac.uk/) are vulnerable to funding cuts depending on the political climate. the unique connection between wikidata and all the wikipedias already make it one of the most well - known and easily discoverable knowledge bases in the world. data deposited here is far less likely to be lost, especially when care is taken to weave it into what already exists. every item loaded to wikidata (mediawiki foundation s third most active project) becomes a fixed point in a stable, self - sustaining knowledge representation platform that anyone can add to, and anyone can help the network grow through sharing the benefits of their own expertise. the open access, community - driven nature of wikidata contributes to its perpetuity, but the major enduring factor is its universal utility. wikidata is a place for knowledge of any conceivable topic from surfing (q159992) to bacteria (q10876). this variety of topics generates community support that a domain - specific, funding - dependent database can not compete with. in addition to support, wikidata creates the ability to link a surfboard (q457689) to surfing (q159992), the sport (p641) it is used in, or c. trachomatis d / uw-3/cx (q20800373) to pelvic inflammatory disease (q558070), a disease it is the cause of (p1542) in humans (q5). moreover, in principle, it allows microbial genetics data to be linked to data from related fields, including pharmacology and epidemiology. these relationship examples highlight another powerful virtue of wikidata compared to other data storage platforms; adding data to wikidata requires the use of meaningful properties for relating entities. it is insufficient to simply state rdf: seealso as the link between two related entities (as many major databases do in their resource description framework ( rdf) (https://www.w3.org/rdf/) representations ). a relationship between items can not be added without an appropriate property in place, requiring the data model to be defined prior to importing the data. this process of creating properties through community discussion and consensus drives the development of their ontology up - front, rather than forcing the burden of integrating ambiguous content downstream to consumers. although wikidata provides an excellent framework for housing some forms of data, it has some limitations. it is a database of referenced claims about the world and should not, for example, be a repository for sequences or raw expression data. there is no built - in reasoning in wikidata. unlike other semantic web implementations, e.g. those built using the owl ontology language, the current wikidata system can not be used to infer new facts nor to automatically cdisrupted by edits from both well - intentioned editors and, at least theoretically, by malicious users (though true vandalism has thus far not happened at detectable levels). in practice, system - level limitations on heck the logical consistency of statements. editors can not be constrained from making claims that may break data models spread across multiple items. as an openly editable resource, it is possible for data to be data integrity checking are dealt with through the operation of user - generated bots that patrol wikidata seeking and correcting problems as they arise. in terms of inferring new facts based on what is represented in wikidata, it is possible to load all of its contents into external implementations that support this kind of reasoning via frequently updated exports of the entire database (https://www.wikidata.org/wiki/wikidata : data base_download). apart from limits on what content can be stored and what operations can be applied to that content, wikidata is also constrained in terms of user interface. when users browse items online at https://wikidata.org, they are seeing one interface for viewing and editing items ranging from protein domains to dutch history museums. while it is possible to use this interface to find useful information, e.g. all the connections that form the network of knowledge depicted in figure 1 can be clicked through, it is certainly not the most ideal human interface for this task. for developers, the sparql query interface provides the most effective way to access data. for end - users, these applications will support both effective information retrieval and visualization, but can also support direct curation of the content by domain experts. even in consideration of these limits, wikidata is a tremendous potential platform for managing the process of collaboratively understanding microbial genomics. in support of this objective, our immediate next steps are to load the remaining 99 microbial reference genomes from ncbi (http://www.ncbi.nlm.nih.gov/genome/browse/reference/), encompassing bacteria that are the most studied and relevant to human health. these items will form a foundation upon which we invite the microbial research community to collaboratively synthesize their knowledge as it evolves into the future. in addition to loading this data, we are collaborating on the development of an extension to the web apollo genome browser that will allow genome annotations from wikidata to be viewed and edited directly within this popular tool. we see this as the first of many bioinformatics applications that can be enhanced by wikidata content and can enable expert users to contribute to its collaborative curation. we invite and encourage the rest of the scientific community to join our cause in creating this universal microbial genomics resource. we have shown that the aggregation of a subset of data enables powerful queries that demonstrate the potential of connecting data from fragmented sources into a centralized, well - defined structure. in addition to data that can be collected from structured data sources and aggregated in wikidata by bot, a great deal of important information resides only in primary literature. it is thus imperative that we engage the microbial research community to help build wikidata to its potential; a centralized, semantic web compatible mechanism for making sense of mountains of microbial genetic data. this work is supported by the national institutes of health under grant gm089820, gm083924, gm114833 and da036134.

the last 20 years of advancement in sequencing technologies have led to sequencing thousands of microbial genomes, creating mountains of genetic data. while efficiency in generating the data improves almost daily, applying meaningful relationships between taxonomic and genetic entities on this scale requires a structured and integrative approach. currently, knowledge is distributed across a fragmented landscape of resources from government - funded institutions such as national center for biotechnology information (ncbi) and uniprot to topic - focused databases like the odb3 database of prokaryotic operons, to the supplemental table of a primary publication. a major drawback to large scale, expert - curated databases is the expense of maintaining and extending them over time. no entity apart from a major institution with stable long - term funding can consider this, and their scope is limited considering the magnitude of microbial data being generated daily. wikidata is an openly editable, semantic web compatible framework for knowledge representation. it is a project of the wikimedia foundation and offers knowledge integration capabilities ideally suited to the challenge of representing the exploding body of information about microbial genomics. we are developing a microbial specific data model, based on wikidata s semantic web compatibility, which represents bacterial species, strains and the gene and gene products that define them. currently, we have loaded 43 694 gene and 37 966 protein items for 21 species of bacteria, including the human pathogenic bacteria chlamydia trachomatis. using this pathogen as an example , we explore complex interactions between the pathogen, its host, associated genes, other microbes, disease and drugs using the wikidata sparql endpoint. in our next phase of development , we will add another 99 bacterial genomes and their gene and gene products, totaling 900,000 additional entities. this aggregation of knowledge will be a platform for community - driven collaboration, allowing the networking of microbial genetic data through the sharing of knowledge by both the data and domain expert.

visual field assessment is a valuable test in the neuro - ophthalmology clinic for determining presence of visual field deficit, aiding localisation of pathological lesion, and for recording improvement, stabilization, or deterioration of the underlying condition. static perimetry is often undertaken with the humphrey automated perimeter (humphrey instruments, dublin, ca), whilst kinetic perimetry has most commonly been undertaken using the goldmann manual perimeter (haag streit, switzerland). both options, when directly compared, have been shown to reliably detect visual field loss. central static programmes such as the sita 30 - 2 strategy have been used most with humphrey perimetry in these studies. however, there is less information regarding the use of peripheral static programmes using humphrey perimetry. semiautomated kinetic perimetry (skp) has been further developed in recent years, most notably with the octopus 900 perimeter (haag streit, switzerland). assessment of the peripheral visual field in addition to assessment of the central field is often required in the evaluation of patients attending neuro - ophthalmology clinics. however, there is limited information available on the comparison of different peripheral visual field programmes. thus, the purpose of this study was to directly compare skp using the octopus 900 perimeter to a peripheral static programme using the humphrey automated perimeter in a neuro - ophthalmology clinic. a prospective cross - section study was undertaken with local ethical approval and in accordance with the tenets of the declaration of helsinki. participants were not preselected for the study but were identified randomly; that is, notes were taken consecutively from the list waiting for visual field assessment without prior knowledge of patient ability and cognition. a selection bias existed in that the patients recruited to this study had been booked to an out - patient neuro - ophthalmology clinic for perimetry. thus, there was an assumption that these patients had sufficient ability and cognition to undertake standard automated perimetry. inclusion criteria were adult patients aged 18 years or older attending for visual field assessment, sufficient motor ability to sit at the perimeter unaided, able to press the response button, sufficient cognitive ability to understand and follow instructions for performing the test, willingness to undertake testing on both perimeters on the same day, and able to respond to both i4e and i2e target stimuli on octopus perimetry. the exclusion criteria were patients with visual acuity less than 1.0 logmar, those unable to sit for the duration of perimetry assessment, follow instructions for performing the test, or too ill to complete the full assessment. all patients underwent perimetry following full explanation of the purpose of the test and procedure. the full field 120 (ff120) two zone programme was used as the peripheral static programme on the humphrey perimeter. this programme consists of 120 stimulus locations with a higher density of locations in the nasal than temporal visual field. for the purposes of standardisation and comparison in this study, a screening protocol was used for skp. two stimuli of the same size (0.25 mm) were used but of different intensity (i4e, 1000 apostilbs and i2e, 100 apostilbs). the peripheral visual field boundary and blind spot were assessed using a size i4e target. a minimum of twelve vectors were assessed for the peripheral visual field and eight for the central visual field inclusive of vectors offset from the vertical and horizontal meridia moving centripetally, similar to previously reported testing strategies. following assessment, the response points along each vector were joined to form the isopter for i4e and i2e targets, respectively. in addition, 56 static points (14 per quadrant) were assessed within the central 30 degrees of the visual field using the i4e target (figure 1). movement of the target on the octopus perimeter was set at 5/sec for determination of central and peripheral isopter boundaries and at 3/sec for determination of the blind spot boundary and quantification of boundaries of visual field defects. the study protocol consisted of visual field assessment with both humphrey and octopus perimetry on the same day. the order of testing was randomised as to which of the two assessment types was used first in order to take fatigue effect into consideration. patients were assigned to one perimeter or another according to which perimeter was available for use at the time the patient was called for assessment. reliability was determined automatically by fixation loss and false positive and false negative responses on humphrey perimetry and by manually checking false positive and false negative responses on octopus perimetry. poor reliability was deemed present with fixation losses and false positive and false negative responses of > 25%. visual field in both groups were assessed for presence or absence of visual field defects. full (normal) visual fields by kinetic assessment were defined as visual field with isopters for i4e and i2e falling within age - matched ranges and no focal defects within the isopter area (apart from the blind spot in the temporal field). visual field loss was defined as isopter boundaries constricted within the age - matched ranges which could be global constriction or a defect type. defect types were classified according to a modified list based on those reported by pineles et al. and the ocular hypertension treatment study (ohts) and outlined in table 1. we added a category of functional visual field loss where the visual field defect followed a tubular or spiral pattern on testing. one author assessed the of octopus perimetry (fr) and the second author assessed the of humphrey perimetry (cn). each reviewer was masked to patient identifiers and to the classification by the other reviewer. further independent assessment of a sample of visual field (n = 36) was made by the third author (mm) who determined whether the paired humphrey and octopus were a match or not. a direct comparison was made for octopus and humphrey perimetry using the statistical package spss version 19 (ibm spss statistics, usa). bland - altman strategy was used to compare the differences between two independent measurements for duration of test versus the average test duration. when analysing the bland - altman , we expected most of the differences to lie within 1.96 sd if normally distributed. provided the differences within 1.96 sd would not be clinically important, we considered that the two methods can be used interchangeably. we therefore set a clinical cutoff of within 1 minute as a clinically acceptable difference between perimeter test durations. the chi - square test was used to evaluate correlation between detection of normal and abnormal test by either perimeter. kappa evaluation of agreement was used to correct the proportion of agreement between perimeters due to chance when evaluating intraobserver interpretation of visual field . a value of 1 was defined as perfect agreement, and a value of > 0.7 was deemed a strong agreement. sixty - nine patients attending neuro - ophthalmology clinics underwent dual testing with humphrey ff120 and octopus skp perimetry during the same clinic visit. five patients were subsequently excluded due to octopus perimetry being undertaken using a size iii4e target or i4e target only. sixty - four remaining patients (113 eyes) had diagnoses of posterior visual pathway pathology, anterior visual pathway pathology, functional impairment of visual field, and thirty - one patients had diagnoses that were classed as neurological defects (postchiasmal) and 28 patients that were classed as ocular defects (prechiasmal). five patients had normal visual fields or nonspecific visual field defects classed as functional or spurious. there were 29 females and 35 males with a mean age of 48 years (sd 14). all patients were able to respond to both i4e and i2e target stimuli. on humphrey perimetry the central and peripheral reference decibel level was a mean of 32.6 (sd 2.4). the mean duration of assessment for skp was 4.54 minutes 0.18 compared to the mean duration for humphrey perimetry of 6.17 0.12 which was significantly different (p = 0.0001 unpaired t test). although the mean duration was higher for humphrey perimetry (difference between means of 1.63 0.22), bland altman analysis showed proportional change when the differences were compared between the two perimeters (figure 2). the confidence intervals ranged from 5.25 to 2.01 minutes with differences exceeding our clinical cutoff of within 1 minute. with larger variances, skp showed longer test durations than humphrey perimetry (16 eyes ( 15%), table 3 ). 80% of (90 eyes) were correctly matched for normal or abnormal visual fields using the i4e target versus humphrey ff120 (table 4), and 73.5% (83 eyes), correctly matched using the i2e target versus humphrey ff120 (table 5). mismatch was due to the i4e isopter being classed as normal or showing only a partial defect or different defect to humphrey perimetry. mismatch with the i2e target was due to the isopter being classed as normal, showing a different defect, partial defect, or being more constricted. in three eyes only, the humphrey was classed as normal while the octopus was classed as showing a visual field defect. in all other discrepancies, the humphrey was worse. when comparing the octopus field with combined i4e and i2e isopters (i.e., either or both targets detecting a defect) to the humphrey , a match for normal or abnormal fields was recorded in 87% (98 eyes). three eyes (2.6%) had normal humphrey and abnormal octopus , while three eyes (2.6%) had normal octopus and abnormal humphrey . the features of these match discrepancies are outlined in table 6. on independent grading of a sample of , 80% of (28 eyes) were correctly matched for normal or abnormal visual fields using the i4e target versus humphrey ff120 (table 7), and 80% (28 eyes) were correctly matched using the i2e target versus humphrey ff120 (table 8). when comparing the octopus field with combined i4e and i2e isopters to the humphrey , a match for normal or abnormal fields was recorded in 83% (29 eyes). the agreement between matching by the first two authors versus independent matching by the third author was significant (p = 0.0001) with 30 of 35 being correctly matched (= 0.8). of the five not correctly matched, each had been matched as abnormal for humphrey and octopus perimetry by the first authors but with a mismatch (humphrey normal and octopus abnormal in four , humphrey abnormal and octopus normal in two ) by the third author. for all comparisons, the humphrey was classed as showing a worse field (greater size of field defect) in 38 eyes. conversely, the octopus was classed as showing a worse field in 20 eyes which was significantly less than humphrey perimetry, p = 0.001 (test). there was no significant difference for humphrey or octopus being worse in abnormal visual field due to either ocular or neurological causes (p = 0.77 and p = 0.964, respectively, test). during humphrey ff120 perimetry there is an initial determination of central and peripheral threshold levels (calculated in decibel values : db) at the beginning of the test. this is used to determine the individual's reference hill of vision and stimuli are subsequently presented at the predetermined test locations at six decibel intensities higher than the expected threshold for each location. a mean reference level of 32.6 db (sd 2.4) was calculated for the humphrey in this study. thus, stimuli intensities would range from a mean of approximately 26 db. for octopus skp we used the i4e target to determine the peripheral boundary of the visual field and i2e for the central boundary. calibration of the octopus with a luminance of 31.4 apostilbs and 1000 apostilb maximum stimulus luminance in a db value of 20 for the i4e target and 30 db for the i2e target. notably, the decibel scale is not standardised across the humphrey and octopus perimeters as the maximum luminance varies between the two perimeters. on comparison of , a correct match of visual field was 80% for the i4e target and 73.5% for the i2e target on skp in comparison to the visual field on humphrey perimetry. furthermore, when both i4e and i2e isopters were compared, in conjunction with each other, to the humphrey , a correct match was recorded for 87% of . thus, combined assessment of the peripheral and central field with octopus perimetry led to the more sensitive detection of visual field deficit eight percent of comparisons both showed abnormal visual field but lacking an accurate match of defect with abnormalities mainly relating to constriction of field versus a defect in different quadrants of the visual field. three eyes (2.6%) had normal humphrey but corresponding octopus showed peripheral superior defects (all in cases of pituitary adenoma). in a further three eyes (2.6%) normal octopus were recorded but humphrey showed abnormalities. it should be noted that in two , the humphrey showed involvement of spurious points or generalised constriction but in which no specific diagnosis of visual field type could be made. thus, these could represent normal visual fields in which the patient failed to make adequate responses to stimuli from time to time. given these comparisons of mismatch , we did not feel that the octopus missed more defects than humphrey, or vice versa. independent grading of a sample of visual field provided similar match comparisons, and a strong agreement for matched by the first two authors in comparison to the third author was found. previous comparative studies have contrasted skp with static perimetry within the central 30 degrees in ocular diseases such as advanced glaucoma, optic neuritis, and optic nerve head drusen, with good comparisons and test - retest reliability. furthermore, improved defection of visual field loss was obtained when both tests were used in conjunction with each other. similar comparisons for neuro - ophthalmic cases have been reported with equal reliability in 77% of eyes, and our are similar to these with skp compared to humphrey ff120 peripheral static perimetry. disadvantages of static perimetry have been reported as inaccurate location of lesion to the anterior visual pathway, failure to detect macular sparing hemianopia, and overestimation of visual field extent. all matched visual field , the humphrey were graded as being more extensive than octopus in 38 eyes, and the octopus were graded as being more extensive than humphrey in 20 eyes. the difference of humphrey being more extensive than octopus was significant and might reflect the presence of statokinetic dissociation which has been defined as the static defect being larger than the kinetic defect. statokinetic dissociation has been reported as occurring as a physiological phenomenon and has been found to increase towards the periphery of the visual field and decrease towards the centre of the visual field. we did not find statokinetic dissociation to be more prevalent in neurological versus ocular causes of visual field loss. a previous comparison of semi - kinetic perimetry versus automated central static threshold perimetry reported a median test duration of 13 minutes for the kinetic option and 11 minutes for the static option. we found the opposite in our study with a mean test duration of 4.54 minutes for skp and 6.17 minutes for humphrey static perimetry. this may reflect the different number of isopters and vectors assessed for octopus perimetry but also the use of a peripheral suprathreshold static programme rather than a central threshold static programme. on further evaluation of the individual test durations versus the average test duration , there was a wide variability, and the humphrey test was not consistently longer than the octopus test. although we used a screening assessment for octopus perimetry to standardise the initial outline of the visual field, we added more vectors to further define visual field defect boundaries (as described in the methods). the skp screening assessment also incorporated static assessment of the visual field similar to previous studies using goldmann perimetry with armaly - drance style strategies. thus, the octopus test became more detailed in the presence of more complex visual field defects. this may explain the crossover of test duration evident on bland - altman analysis in which the octopus test duration was longer than the humphrey test duration in 16 eyes (15%). the humphrey ff120 programme utilised a two zone strategy in which stimuli were recorded as either seen or unseen and would not provide any detailed information with respect to the depth of visual field defect. in these cases, it could be argued that octopus perimetry provides more detailed and informative evaluation of the visual field with better representation of the field defect in terms of its relative or absolute defect severity and which may be more representative of the individuals field defect that was shown by automated perimetry. further evaluation of this aspect in conjunction with patient reported outcome measures for impact of visual field on activities of daily living and quality of life would be useful. although the cases recruited to this study were representative of the types of pathology and visual field defects seen in our neuro - ophthalmology clinics, a larger sample of posterior versus anterior visual pathway defects would have allowed greater comparisons of differences between skp versus static perimetry. our comparison of 28 patients with ocular pathology to 31 patients with neurological pathology did not show any significant differences. a comparison of the ff120 peripheral strategy to a central threshold strategy would be useful to determine if the central threshold static programmes indicate the presence of visual field defects that may impinge more on peripheral than central visual field, such as in cases of pituitary adenoma. this study demonstrates that the combined octopus i4e and i2e targets were more sensitive to detection of visual field loss than either target alone. generally humphrey perimetry test duration was longer than octopus skp although this was not consistent for all tests. when a more detailed evaluation with octopus skp was undertaken, this was at times longer than the humphrey assessment. in the absence of kinetic perimetry options in neuro - ophthalmology clinics, peripheral static suprathreshold programme options such as octopus semi - kinetic perimetry utilising both the i4e and i2e targets provides detailed information of both the defect depth and size and may provide a more representative view of the actual visual field defect, particularly for more moderate to severe visual field defects.

aim. to compare semikinetic perimetry (skp) on octopus 900 perimetry to a peripheral static programme with humphrey automated perimetry. methods. prospective cross - section study comparing humphrey full field (ff) 120 two zone programme to a screening protocol for skp on octopus perimetry. were independently graded for presence / absence of field defect plus type and location of defect. . 64 patients (113 eyes) underwent dual perimetry assessment. mean duration of assessment for skp was 4.54 minutes 0.18 and 6.17 0.12 for ff120 (p = 0.0001). 80% of were correctly matched for normal or abnormal visual fields using the i4e target versus ff120, and 73.5% were correctly matched using the i2e target versus ff120. when comparing octopus with combined i4e and i2e isopters to the ff120 , a match for normal or abnormal fields was recorded in 87%. . humphrey perimetry test duration was generally longer than octopus skp. in the absence of kinetic perimetry , peripheral static suprathreshold programme options such as ff120 may be useful for detection of visual field defects. however, statokinetic dissociation may occur. octopus skp utilising both i4e and i2e targets provides detailed information of both the defect depth and size and may provide a more representative view of the actual visual field defect.

the online version of this article (doi:10.1007/s11032 - 013 - 9929 - 6) contains supplementary material, which is available to authorized users. supplementary material 1 (pdf 29 kb) supplementary material 2 (pdf 87 kb) supplementary material 3 (pdf 299 kb)

high fruit and wine quality combined with good climatic adaptation and disease resistance are essential objectives of grape breeding. while several molecular markers are available for pyramiding resistance to fungal pathogens, molecular tools for predicting fruit composition are still scarce. muscat flavor, caused by the accumulation of monoterpenoids in the berry, is an important target trait for breeding, sought after in both table grapes and wine. four missense mutations in the vvdxs gene in grape germplasm have been shown to be tightly linked to muscat flavor. here we present highly reproducible and breeder - friendly functional markers for each of the targeted polymorphisms developed by using either the multiplexed minisequencing snapshot method, the high - resolution melting (hrm) assay or the cleaved amplified polymorphic sequence system. a total of 242 grapevine accessions were analyzed to optimize these different genotyping methods and to provide allele - specific markers for accurate selection of muscat flavor at early stages of grape breeding programs. the hrm and the minisequencing snapshot multiplex assays allow for high - throughput automated screening and are suitable for large - scale breeding programs and germplasm characterization.electronic supplementary materialthe online version of this article (doi:10.1007/s11032 - 013 - 9929 - 6) contains supplementary material, which is available to authorized users.

aric is a longitudinal study of 15,792 adults aged 4564 years at enrollment in 19871989 in four communities: forsyth county, nc; jackson, ms (african americans only); the northwestern suburbs of minneapolis, mn; and washington county, md. participants attended three subsequent examinations approximately every 3 years (19901992 ; 19931995 ; and 19961999). cardia is a longitudinal study investigating 5,115 african american and white men and women aged 1830 years at enrollment in 19851986 in four communities: birmingham, al; chicago, il; minneapolis, mn; and oakland, ca. participant recruitment was approximately balanced on age, sex, race, and education status at each community. six subsequent examinations were conducted (19871988, 19901991, 19921993, 19951996, 20002001, and 20052006). the offspring cohort of the framingham heart study began in 19711975, enrolling 5,124 offspring and spouses of the offspring of the framingham heart study s original cohort. they were next examined 8 years later and then about every 4 years through the seventh examination, followed by the eighth examination approximately 6.5 years later. initial eligibility criteria for these analyses included participants aged 3554 years old and nondiabetic (defined as fasting blood glucose < 126 mg / dl, and no prior history of and not on medication for diabetes) at their index or baseline examination (as detailed below). this yielded 12,119 participants: 8,170 from aric, 2,111 from cardia, and 1,838 from the framingham heart study. participants were further excluded if they were not african american or white (n = 30); failed to return for follow - up or did not have at least one follow - up visit to determine diabetes status (n = 709); or at the index examination were either pregnant (n = 12), did not fast > 8 h (n = 330), or had missing data on systolic (sbp) or diastolic blood pressure (dbp) or any of the following cardiometabolic traits: fasting blood glucose, insulin, hdl cholesterol (hdl - c), or triglycerides, waist circumference, or bmi (n = 232). those excluded were more likely to be african american (40.5 vs. 23.4%), not finished high school (21.1 vs. 12.4%), have higher sbp (mean 119 vs. 116 mmhg), and prevalence of hypertension (28.1 vs. 21.5%), consume more alcohol (mean 4.8 vs. 3.8 drinks / week), and be a smoker (40.8 vs. 25.2%). for cardia and the framingham heart study offspring cohort, their fifth examination cycle (conducted in 19951996 and 19911995, respectively) was considered the index examination, for an approximate follow - up period of 10 and 14 years, respectively. these were chosen over prior examinations to ensure a more contemporary sample while allowing for sufficient follow - up of approximately 1 decade. because the most recent aric exam occurred in 19961999, its first examination (conducted in 19871989) was considered the index examination, for 9 years of follow - up. blood pressure was measured with participants seated after a 5-minute rest using a random - zero mercury sphygmomanometer in aric and cardia, and a standard mercury - column sphygmomanometer in the framingham heart study. three mutually exclusive blood pressure categories were established: hypertension was defined if sbp 140 mmhg, dbp 90 mmhg, or reported use of antihypertensive medication; prehypertension was defined as not having hypertension, and sbp 120139 mmhg or dbp 8089 mmhg; normal included sbp < 120 mmhg and dbp < 80 mmhg and not using antihypertensive medication. bmi was calculated as weight in kilograms divided by the square of height in meters. waist circumference was measured at the level of the umbilicus in aric and the framingham heart study, and at the level of the smallest waist circumference in cardia. self - reported information included race, education level, parental history of diabetes (one or both parents with diabetes), smoking status, alcohol use, and physical activity. participants were instructed to fast overnight before providing blood specimens for measuring glucose, lipid, and insulin levels. participants were considered to have incident diabetes if any of the following was present at a follow - up examination: fasting blood glucose 126 mg / dl, casual blood glucose 200 mg / dl, or using insulin or oral hypoglycemic medication. time - to - diabetes was estimated using a previously described method by duncan et al. the incident date was estimated by linear interpolation using the glucose values at the ascertaining and previous examinations. for cases ascertained based on the use of diabetic medications, the time - to - diabetes was estimated by using their fasting glucose at the earlier visit and a slope estimated using information from all diabetic subjects who had been unaware of their status (because the fasting glucose at ascertainment for those who were on diabetic medication may have been affected by their knowledge of their diabetes status). general linear models were used to compare characteristics by blood pressure categories after adjustment for age and sex (regression models for continuous traits and poisson regression for categorical traits). in multivariable analyses, age - specific rates were first calculated and then weighted to the standard year 2000 u.s. population to derive age - adjusted incidence rates by race, sex, and blood pressure category. confidence intervals and trend tests across blood pressure category within race and sex groups were estimated using 2,000 bootstrap resamples. a standard normal distribution in the rates was assumed because the bias between the sample population estimates and the mean of the bootstrap estimates was less than 1%. proportional hazards models were fitted to assess the association of blood pressure category with incident diabetes. tests for effect modification by sex were evaluated in race - specific models that adjusted for age and sex, whereas effect modification by race was tested in age-, race-, and sex - adjusted models. nonproportionality of hazards over time was tested in race - specific models using time - dependent covariates for the blood pressure categories. for multivariable analyses, cox proportional hazards models were constructed to calculate the hazard ratio (hr) and 95% cis to compare incident diabetes across blood pressure categories, using normal blood pressure as the reference group. the base model adjusted for age and sex. the third, considered our primary model, further adjusted for fasting glucose, hdl - c, and triglyceride. fasting glucose was included to account for its potential confounding effect on blood pressure and incident diabetes because its levels were positively associated with increasing blood pressure categories in both race groups. the fourth model included a longer list of cardiometabolic risk factors by also introducing fasting insulin and waist circumference. to directly compare the racial differences in the association of blood pressure and incident diabetes , we also combined both races in multivariable analyses with race - specific blood pressure categories using normotensive whites as the referent group. sensitivity analyses included constructing models with additional groups of covariates or varying the exclusion criteria. the additional covariates included: current smoking, alcohol use, class of antihypertensive drugs, and physical activity level, as well as education level and parental history of diabetes. finally, because those with higher blood pressure also tend to have higher fasting glucose level, which is a strong predictor of diabetes, we performed separate multivariable analyses after lowering our exclusion threshold for baseline fasting glucose to 110 mg / dl from 126 mg / dl (i.e., excluding individuals with borderline elevated levels at the index examination). antihypertensive medications were classified into one of four categories: -blockers, thiazides, ace inhibitors or angiotensin - receptor blockers, or other single - agent medications. combination or multiple medications were sorted into nonmutually exclusive categories (for example, someone taking a -blocker and thiazide was included in each of those two drug classes). therefore, each study s physical activity summary score (sum of leisure, sport, and work activity scores in aric and cardia, and total physical activity in kilocalories over the past year in framingham) was standardized (mean = 0, sd = 1) for analytic purposes. to achieve a normal distribution in the framingham physical activity score initial eligibility criteria for these analyses included participants aged 3554 years old and nondiabetic (defined as fasting blood glucose < 126 mg / dl, and no prior history of and not on medication for diabetes) at their index or baseline examination (as detailed below). this yielded 12,119 participants: 8,170 from aric, 2,111 from cardia, and 1,838 from the framingham heart study. participants were further excluded if they were not african american or white (n = 30); failed to return for follow - up or did not have at least one follow - up visit to determine diabetes status (n = 709); or at the index examination were either pregnant (n = 12), did not fast > 8 h (n = 330), or had missing data on systolic (sbp) or diastolic blood pressure (dbp) or any of the following cardiometabolic traits: fasting blood glucose, insulin, hdl cholesterol (hdl - c), or triglycerides, waist circumference, or bmi (n = 232). those excluded were more likely to be african american (40.5 vs. 23.4%), not finished high school (21.1 vs. 12.4%), have higher sbp (mean 119 vs. 116 mmhg), and prevalence of hypertension (28.1 vs. 21.5%), consume more alcohol (mean 4.8 vs. 3.8 drinks / week), and be a smoker (40.8 vs. 25.2%). for cardia and the framingham heart study offspring cohort, their fifth examination cycle (conducted in 19951996 and 19911995, respectively) was considered the index examination, for an approximate follow - up period of 10 and 14 years, respectively. these were chosen over prior examinations to ensure a more contemporary sample while allowing for sufficient follow - up of approximately 1 decade. because the most recent aric exam occurred in 19961999, its first examination (conducted in 19871989) was considered the index examination, for 9 years of follow - up. blood pressure was measured with participants seated after a 5-minute rest using a random - zero mercury sphygmomanometer in aric and cardia, and a standard mercury - column sphygmomanometer in the framingham heart study. three mutually exclusive blood pressure categories were established: hypertension was defined if sbp 140 mmhg, dbp 90 mmhg, or reported use of antihypertensive medication; prehypertension was defined as not having hypertension, and sbp 120139 mmhg or dbp 8089 mmhg; normal included sbp < 120 mmhg and dbp < 80 mmhg and not using antihypertensive medication. bmi was calculated as weight in kilograms divided by the square of height in meters. waist circumference was measured at the level of the umbilicus in aric and the framingham heart study, and at the level of the smallest waist circumference in cardia. self - reported information included race, education level, parental history of diabetes (one or both parents with diabetes), smoking status, alcohol use, and physical activity. participants were instructed to fast overnight before providing blood specimens for measuring glucose, lipid, and insulin levels. participants were considered to have incident diabetes if any of the following was present at a follow - up examination: fasting blood glucose 126 mg / dl, casual blood glucose 200 mg / dl, or using insulin or oral hypoglycemic medication. time - to - diabetes was estimated using a previously described method by duncan et al. the incident date was estimated by linear interpolation using the glucose values at the ascertaining and previous examinations. for cases ascertained based on the use of diabetic medications, the time - to - diabetes was estimated by using their fasting glucose at the earlier visit and a slope estimated using information from all diabetic subjects who had been unaware of their status (because the fasting glucose at ascertainment for those who were on diabetic medication may have been affected by their knowledge of their diabetes status). general linear models were used to compare characteristics by blood pressure categories after adjustment for age and sex (regression models for continuous traits and poisson regression for categorical traits). in multivariable analyses, age - specific rates were first calculated and then weighted to the standard year 2000 u.s. population to derive age - adjusted incidence rates by race, sex, and blood pressure category. confidence intervals and trend tests across blood pressure category within race and sex groups were estimated using 2,000 bootstrap resamples. a standard normal distribution in the rates was assumed because the bias between the sample population estimates and the mean of the bootstrap estimates was less than 1%. proportional hazards models were fitted to assess the association of blood pressure category with incident diabetes. tests for effect modification by sex were evaluated in race - specific models that adjusted for age and sex, whereas effect modification by race was tested in age-, race-, and sex - adjusted models. nonproportionality of hazards over time was tested in race - specific models using time - dependent covariates for the blood pressure categories. for multivariable analyses, cox proportional hazards models were constructed to calculate the hazard ratio (hr) and 95% cis to compare incident diabetes across blood pressure categories, using normal blood pressure as the reference group. the base model adjusted for age and sex. the third, considered our primary model, further adjusted for fasting glucose, hdl - c, and triglyceride. fasting glucose was included to account for its potential confounding effect on blood pressure and incident diabetes because its levels were positively associated with increasing blood pressure categories in both race groups. the fourth model included a longer list of cardiometabolic risk factors by also introducing fasting insulin and waist circumference. to directly compare the racial differences in the association of blood pressure and incident diabetes, we also combined both races in multivariable analyses with race - specific blood pressure categories using normotensive whites as the referent group. sensitivity analyses included constructing models with additional groups of covariates or varying the exclusion criteria. the additional covariates included: current smoking, alcohol use, class of antihypertensive drugs, and physical activity level, as well as education level and parental history of diabetes. finally, because those with higher blood pressure also tend to have higher fasting glucose level, which is a strong predictor of diabetes, we performed separate multivariable analyses after lowering our exclusion threshold for baseline fasting glucose to 110 mg / dl from 126 mg / dl (i.e., excluding individuals with borderline elevated levels at the index examination). antihypertensive medications were classified into one of four categories: -blockers, thiazides, ace inhibitors or angiotensin - receptor blockers, or other single - agent medications. combination or multiple medications were sorted into nonmutually exclusive categories (for example, someone taking a -blocker and thiazide was included in each of those two drug classes). therefore, each study s physical activity summary score (sum of leisure, sport, and work activity scores in aric and cardia, and total physical activity in kilocalories over the past year in framingham) was standardized (mean = 0, sd = 1) for analytic purposes. to achieve a normal distribution in the framingham physical activity score, nearly one - quarter (23.4%) were african americans, all from aric and cardia. mean age (sd) in years in cardia, aric, and framingham heart study were 37.7 (1.72), 49.9 (3.15), and 47.5 (5.00), respectively (supplementary table a). table 1 shows that at baseline, within each race increasingly higher blood pressure categories were significantly associated with lower educational level, hdl - c, and physical activity scores, as well as older age, higher fasting glucose, triglycerides and insulin levels, and greater waist circumference, bmi, and alcohol consumption. higher blood pressure was also associated with higher proportion of men, current smoker, and parental history of diabetes in whites. overall, whites had more favorable profiles in blood pressure, adiposity, fasting glucose and insulin levels, prevalence of smoking, and education, whereas african americans had more favorable lipid levels. age- and sex - adjusted means or proportions of baseline characteristics by race and blood pressure category * at baseline data are mean (sd) or percent, unless otherwise indicated. bp, blood pressure; dm, diabetes; hs, high school; wc, waist circumference. * blood pressure categories: normal, sbp < 120 and dbp < 80 mmhg and not using antihypertensive medication; prehypertension, not hypertension and sbp 120139 or dbp 8089 mmhg; hypertension, sbp 140, dbp 90 mmhg, or using antihypertensive medication. exponent of log transformed triglycerides, therefore is approximately the median of the untransformed distribution. the sd is from the distribution of log (triglycerides). p value from a simple linear contrast. a higher z score means a greater physical activity level of that participant relative to others in the same study. for example, a z score of 1.96 means that that participant was more physically active than 95% of the participants in that study. during median follow - up of 8.9 years, 14.6% (n = 372 ; 239 women) of african americans and 7.9% (n = 657 ; 271 women) of whites developed diabetes. within each race - sex group, age - adjusted rates were increasingly higher across baseline blood pressure categories, with the incidence lowest in the normal blood pressure group and highest in the hypertension group (fig . 1) (p values for trend : < 0.05 for african american men ; < 0.001 for other race - sex groups). the rates ranged from 2.8 per 1,000 person - years in normotensive white women to 28.9 per 1,000 person - years in hypertensive african american women. age - adjusted rate of incident diabetes and 95% ci by baseline blood pressure category, sex, and race. rate per 1,000 person - years, age - adjusted to the year 2000 standard population; 95% ci from normal approximation after 2,000 bootstrap samples. p values for trend all race and sex groups < 0.0001, except for african american men, for which p value for trend was 0.0219. normal blood pressure: sbp < 120 and dbp < 80 mmhg and not using antihypertensive medication; prehypertension: not hypertension and sbp 120139 or dbp 8089 mmhg; and hypertension: sbp 140, dbp 90 mmhg, or using antihypertensive medication. race - stratified multivariable analyses were performed because tests for race - by - blood - pressure - category interactions were significant (p values - for - interaction for prehypertension and hypertension : 0.004 and 0.003, respectively). within race - specific models, effect modification by sex was not present (p values - for - interaction for prehypertension and hypertension : 0.68 and 0.92 in african americans, and 0.37 and 0.052 in whites, respectively); therefore, sex - pooled models were used for all analyses. in models with interaction terms for study - by - blood - pressure - category, the age- and sex - adjusted hr for diabetes among african americans (table 2) with hypertension was 1.95 (95% ci 1.502.54), using normal blood pressure group as the referent; the risk was not significant for prehypertension (p = 0.23). after further adjusting for bmi, the diabetes risk associated with hypertension was attenuated but remained statistically significant (hr 1.48); but when fasting glucose, hdl - c, and triglyceride levels were added, the effect was further attenuated and there was no longer a significant association (0.92). hrs for the association of baseline prehypertension and hypertension with incident diabetes * using normal blood pressure (sbp < 120 and dbp < 80 mmhg and not using antihypertensive medication) as referent for comparing prehypertension (not hypertension and sbp 120139 or dbp 8089 mmhg) and hypertension (sbp diabetes during follow - up defined as fasting glucose 126 mg / dl, casual glucose 200 mg / dl, or use of diabetic drugs . model 2 + fasting glucose, hdl, and triglycerides . among whites ( table 2), after adjusting for age and sex, compared with normal blood pressure group, hypertension had a more than threefold increased risk of developing diabetes (hr 3.26), and prehypertension had a twofold increased risk (1.99). these associations remained statistically significant, though attenuated, after also adjusting for bmi, fasting glucose, hdl - c, and triglyceride levels (hr for prehypertension : 1.32 ; for hypertension : 1.25). the race - specific remained similar even when the modeling included additional baseline covariates (first, waist circumference and fasting insulin ; then also antihypertensive drug class, smoking, alcohol, and physical activity ; and finally also education level and parental history of diabetes). statistically significant associations between prehypertension or hypertension and incident diabetes were again observed in whites only (supplementary table b). of note, in the multivariable analyses no antihypertensive drug class was significantly associated with incident diabetes ( not shown). in race - combined primary multivariable model (i.e., adjusting for age, sex, bmi, fasting glucose, hdl - c, and triglyceride) using normotensive whites as referent, the hr (95% ci) for developing diabetes in normotensive, prehypertensive, and hypertensive african americans were 2.75 (2.143.53), 2.28 (1.762.95), and 2.36 (1.932.90), respectively, and in prehypertensive and hypertensive whites were 1.36 (1.121.65) and 1.33 (1.101.62), respectively. a similar pattern was observed after additionally adjusting for other differences in baseline characteristics: education, fasting insulin, waist circumference, smoking, alcohol, and parental history of diabetes ( not shown). when repeating our primary multivariable model after lowering the baseline exclusion glucose cut point to 110 mg / dl from 126 mg / dl, the significant associations persisted among whites (hr for prehypertension : 1.42 ; hr for hypertension : 1.52). in african americans , there was now a trend toward higher risk of diabetes in prehypertension group (1.18) and a marginally statistically significant association in hypertension group (1.44), which was no longer significant after additionally adjusting for fasting insulin and waist circumference (p value 0.062). in a race - pooled multivariable model with baseline glucose < 110 mg / dl and using normotensive whites as referent, the race - by - blood - pressure - category interaction terms were not significant (p values > 0.395). nearly one - quarter (23.4%) were african americans, all from aric and cardia. mean age (sd) in years in cardia, aric, and framingham heart study were 37.7 (1.72), 49.9 (3.15), and 47.5 (5.00), respectively (supplementary table a). table 1 shows that at baseline, within each race increasingly higher blood pressure categories were significantly associated with lower educational level, hdl - c, and physical activity scores, as well as older age, higher fasting glucose, triglycerides and insulin levels, and greater waist circumference, bmi, and alcohol consumption. higher blood pressure was also associated with higher proportion of men, current smoker, and parental history of diabetes in whites. overall, whites had more favorable profiles in blood pressure, adiposity, fasting glucose and insulin levels, prevalence of smoking, and education, whereas african americans had more favorable lipid levels. age- and sex - adjusted means or proportions of baseline characteristics by race and blood pressure category * at baseline data are mean (sd) or percent, unless otherwise indicated. bp, blood pressure; dm, diabetes; hs, high school; wc, waist circumference. * blood pressure categories: normal, sbp < 120 and dbp < 80 mmhg and not using antihypertensive medication; prehypertension, not hypertension and sbp 120139 or dbp 8089 mmhg; hypertension, sbp 140, dbp 90 mmhg, or using antihypertensive medication. exponent of log transformed triglycerides, therefore is approximately the median of the untransformed distribution. the sd is from the distribution of log (triglycerides). p value from a simple linear contrast. a higher z score means a greater physical activity level of that participant relative to others in the same study. for example, a z score of 1.96 means that that participant was more physically active than 95% of the participants in that study. during median follow - up of 8.9 years, 14.6% (n = 372 ; 239 women) of african americans and 7.9% (n = 657 ; 271 women) of whites developed diabetes. within each race - sex group, age - adjusted rates were increasingly higher across baseline blood pressure categories, with the incidence lowest in the normal blood pressure group and highest in the hypertension group (fig . 1) (p values for trend : < 0.05 for african american men ; < 0.001 for other race - sex groups). the rates ranged from 2.8 per 1,000 person - years in normotensive white women to 28.9 per 1,000 person - years in hypertensive african american women. age - adjusted rate of incident diabetes and 95% ci by baseline blood pressure category, sex, and race. rate per 1,000 person - years, age - adjusted to the year 2000 standard population; 95% ci from normal approximation after 2,000 bootstrap samples. p values for trend all race and sex groups < 0.0001, except for african american men, for which p value for trend was 0.0219. normal blood pressure: sbp < 120 and dbp < 80 mmhg and not using antihypertensive medication; prehypertension: not hypertension and sbp 120139 or dbp 8089 mmhg; and hypertension: sbp 140, dbp 90 mmhg, or using antihypertensive medication. race - stratified multivariable analyses were performed because tests for race - by - blood - pressure - category interactions were significant (p values - for - interaction for prehypertension and hypertension : 0.004 and 0.003, respectively). within race - specific models, effect modification by sex was not present (p values - for - interaction for prehypertension and hypertension : 0.68 and 0.92 in african americans, and 0.37 and 0.052 in whites, respectively); therefore, sex - pooled models were used for all analyses. in models with interaction terms for study - by - blood - pressure - category, the age- and sex - adjusted hr for diabetes among african americans (table 2) with hypertension was 1.95 (95% ci 1.502.54), using normal blood pressure group as the referent; the risk was not significant for prehypertension (p = 0.23). after further adjusting for bmi, the diabetes risk associated with hypertension was attenuated but remained statistically significant (hr 1.48); but when fasting glucose, hdl - c, and triglyceride levels were added, the effect was further attenuated and there was no longer a significant association (0.92). hrs for the association of baseline prehypertension and hypertension with incident diabetes * using normal blood pressure (sbp < 120 and dbp < 80 mmhg and not using antihypertensive medication) as referent for comparing prehypertension (not hypertension and sbp 120139 or dbp 8089 mmhg) and hypertension (sbp diabetes during follow - up defined as fasting glucose 126 mg / dl, casual glucose 200 mg / dl, or use of diabetic drugs . model 2 + fasting glucose, hdl, and triglycerides . among whites ( table 2), after adjusting for age and sex, compared with normal blood pressure group, hypertension had a more than threefold increased risk of developing diabetes (hr 3.26), and prehypertension had a twofold increased risk (1.99). these associations remained statistically significant, though attenuated, after also adjusting for bmi, fasting glucose, hdl - c, and triglyceride levels (hr for prehypertension : 1.32 ; for hypertension : 1.25). the race - specific remained similar even when the modeling included additional baseline covariates (first, waist circumference and fasting insulin ; then also antihypertensive drug class, smoking, alcohol, and physical activity ; and finally also education level and parental history of diabetes). statistically significant associations between prehypertension or hypertension and incident diabetes were again observed in whites only (supplementary table b). of note, in the multivariable analyses no antihypertensive drug class was significantly associated with incident diabetes ( not shown). in race - combined primary multivariable model (i.e., adjusting for age, sex, bmi, fasting glucose, hdl - c, and triglyceride) using normotensive whites as referent, the hr (95% ci) for developing diabetes in normotensive, prehypertensive, and hypertensive african americans were 2.75 (2.143.53), 2.28 (1.762.95), and 2.36 (1.932.90), respectively, and in prehypertensive and hypertensive whites were 1.36 (1.121.65) and 1.33 (1.101.62), respectively. a similar pattern was observed after additionally adjusting for other differences in baseline characteristics: education, fasting insulin, waist circumference, smoking, alcohol, and parental history of diabetes ( not shown). when repeating our primary multivariable model after lowering the baseline exclusion glucose cut point to 110 mg / dl from 126 mg / dl, the significant associations persisted among whites (hr for prehypertension : 1.42 ; hr for hypertension : 1.52). in african americans, there was now a trend toward higher risk of diabetes in prehypertension group (1.18) and a marginally statistically significant association in hypertension group (1.44), which was no longer significant after additionally adjusting for fasting insulin and waist circumference (p value 0.062). in a race - pooled multivariable model with baseline glucose < 110 mg / dl and using normotensive whites as referent, the race - by - blood - pressure - category interaction terms were not significant (p values > 0.395). middle - aged african americans and whites with higher blood pressure are more likely to develop diabetes than those with normal blood pressure. in african americans, the higher incidence of diabetes among hypertensive individuals may be explained by concomitantly greater adiposity and other cardiometabolic risk factors. in whites, the association of both prehypertension and hypertension with incident diabetes regardless of baseline blood pressure status, african americans have a greater risk of developing diabetes than whites. only one prior aric publication from a decade ago included substantial numbers of african americans when studying the relationship between blood pressure and new - onset diabetes; however, race - specific were not presented. we found significant race - by - blood - pressure - category interaction for incident diabetes in our primary analysis, thus needing separate analyses by race. although after accounting for age- and sex - differences hypertensive african americans were more than twice as likely to develop diabetes as normotensive african americans, after further adjusting for baseline bmi, fasting glucose, hdl - c, and triglyceride levels, the effect size was greatly attenuated and no longer significant. for whites, our finding validates prior reports that high blood pressure is a risk factor for diabetes. this finding is in contrast to a recent paper from the san antonio heart study, where no association between prehypertension and diabetes was observed in 2,767 mexican americans and non - hispanic white participants after multivariable analysis. although their age-, sex-, and race - adjusted model of prehypertension was associated with incident diabetes, it was no longer significant after further adjusting for bmi, impaired glucose tolerance, insulin resistance and secretion, and family history. however, the multivariable - adjusted odds ratio of incident diabetes for prehypertension versus normal blood pressure at 1.42 (95% ci 0.992.02) was comparable to that of our prehypertensive whites. african americans generally have higher levels of cardiovascular risk factors than whites except for lipid profile. even after we accounted for these differences, the racial variation in the association between blood pressure and incident diabetes persisted. however, when lowering the exclusion - criteria threshold for baseline fasting glucose to 110 mg / dl, the positive in whites remained robust, whereas trends toward positive associations were newly observed in african americans. in a race - pooled model with this lower exclusion cutpoint and using normotensive whites as referent, the race - by - blood pressure - category interaction terms also became no longer significant. taken together, this suggests that the null association from our primary analyses of african americans may have been largely driven by their higher overall baseline glucose levels. although african americans are known to have greater risks of developing diabetes than whites, potentially modifiable factors including obesity, education, physical inactivity, smoking, alcohol, and dietary intake have accounted for only about half of the disparity. we further found that this disparity can not be explained by african americans higher prevalence of prehypertension and hypertension. even after accounting for racial differences in several risk factors, their risk of diabetes remained higher than whites across all blood pressure categories. notably, education is a limited proxy for socioeconomic status, and other measures such as income and healthcare access are needed. higher fasting insulin levels may be one potential biological mechanism to explain the excess incidence of diabetes, though only in nonobese african american women. future studies should investigate additional factors such as insulin sensitivity, inflammation, and endothelial dysfunction, the pathways of which, like insulin resistance, are shared by hypertension and diabetes. strengths of the present article include the large sample size, multiethnic sample, measured instead of self - reported blood pressure and blood glucose; and extensive data on potential confounders. first, as with all observational studies, residual confounding can not be ruled out. second, the african american participants were only from aric and cardia. in aric, > 90% of the african americans were recruited from one site (jackson, ms). given the nonoverlapping age - range between aric and cardia participants, we did not test whether african americans differ between these studies. though possible that the observed race - and - blood pressure interaction might have been confounded by racial differences in geographical distribution, cardia included balanced biracial cohorts from four other cities, making this concern less likely. third, whether causal relationship exists and whether treating high blood pressure prevents diabetes are outside the scope of observational studies. trials on prehypertension are few and none reported incident diabetes as an outcome. for hypertension, several meta - analyses of antihypertensive drug trials that reported incident diabetes identified inhibitors of the renin - angiotensin system to lower the risk. in contrast, other antihypertensive drugs such as -blockers and diuretics were implicated as putative risk - promoting factors. moreover, drug trials have been inherently limited in their inability to distinguish any protective effects of blood pressure - lowering per se from any direct pharmacological effects on diabetes development. in summary, high blood pressure in middle age is associated with greater likelihood of developing diabetes. in african americans with hypertension, this association both prehypertension and hypertension are associated with increased risk of developing diabetes beyond that explained by adiposity and other risk factors. whether lowering blood pressure slows or prevents the onset of diabetes deserves further clinical investigation. regardless of blood pressure status, african americans have greater risks of developing diabetes than whites. future studies are also needed to determine the etiology for the excess risks in african americans.

objectivewe examined the association between high blood pressure and incident type 2 diabetes in african americans and whites aged 3554 years at baseline.research design and methodswe combined data from the atherosclerosis risk in communities (aric) study, the coronary artery risk development in young adults (cardia) study, and the framingham heart study offspring cohort. overall, 10,893 participants (57% women ; 23% african american) were categorized by baseline blood pressure (normal, prehypertension, hypertension) and examined for incident diabetes (median follow - up 8.9 years).overall, 14.6% of african americans and 7.9% of whites developed diabetes. age - adjusted incidence was increasingly higher across increasing blood pressure groups (p values for trend : < 0.05 for african american men ; < 0.001 for other race - sex groups). after adjustment for age, sex, bmi, fasting glucose, hdl cholesterol, and triglycerides, prehypertension or hypertension (compared with normal blood pressure) was associated with greater risks of diabetes in whites (hazard ratio for prehypertension : 1.32 ; for hypertension : 1.25), but not african americans (hr for prehypertension : 0.86 ; for hypertension : 0.92). hrs for developing diabetes among normotensive, prehypertensive, and hypertensive african americans versus normotensive whites were: 2.75, 2.28, and 2.36, respectively (p values < 0.001).in african americans, higher diabetes incidence among hypertensive individuals may be explained by bmi, fasting glucose, triglyceride, and hdl cholesterol. in whites, prehypertension and hypertension are associated with greater risk of diabetes, beyond that explained by other risk factors. african americans, regardless of blood pressure, have greater risks of developing diabetes than whites.

empowerment of women and improvement of their conditions and positions in the society are the salient issues to achieve mental health. body image is one of the important psychological concepts, which has always been a concern for men and women. body image is a subjective concept and one of the important concepts of self - image. this image is a personal relationship that everyone has with his / her own body, in addition to a set of beliefs, perceptions, thoughts, feelings, and actions, which are related to the physical appearance of humans. physical conception is rooted in scientific psychology and is a proactive, cognitive, and behavioral approach. based on the cognitive behavioral theory of cash, the influencing factors on body image are derived from individual, environmental and behavioral factors. the events that occurred in the past and present for each person are also involved in the formation of body image. the dissatisfaction of many women of their bodies from natural changes in the body such as genetic structure of the body, short stature in women compared with men, maturity, rapid growth of the body in adolescence, induced menstrual periods, process of aging, and other things that change (short term or long term) the body image and self - concept of the individual. one of the major changes in women's lives is pregnancy and childbirth, which is enjoyable for many women. therefore, childbearing can be an opportunity to reduce their sensitivity to the attraction and beauty of their bodies, which leads to a period of tranquillity and relaxation about their body images. however, women who do not experience pregnancy may feel that their bodies do not work properly. one of the disturbing difficulties of women is infertility or the inability of the woman or man to have children. brunner's findings indicated that the diseases cause loss of control over the body, lead to the feeling of inadequacy in a person from the body, and create insecurity and negative body image. for infertile women, the inability to become pregnant and experience childbirth is considered as a kind of impairment caused by acquired, inherited, or congenital causes. however, infertility is not a disease, but it can cause major affective disorders. when married women do not have children until the age of 30, they discover that they may have fertility problem and it is possible to find negative body image in them because they understand that they do not have the ability for childbearing. on the other hand, in infertile women with a poor body image, more psychological problems can be observed. for example, this can be a cause of concern for sexual and physical attractiveness, general feeling of self - esteem, and general health. meanwhile, these negative emotions are uninformed, but women still do combat with this issue mentally. infertile women are deprived of pregnancy relaxation periods, and this issue is important from their psychopathological point of view. in the study of younesi and salajegheh, it has been reported that infertile women had more problems about body image compared with fertile women. in contrast, khodakarami et al. have reported that women may pay less attention to their physical appearances, beauty, and body image due to the fear of an unknown future, the possibility of failure in the treatment of infertility and its consequences. for example, a direct correlation has been found between feeding behavior and body image. therefore, body image may impact behaviors such as sexual function too, because it is associated with the physical aspects- (physical and sexual attraction) and also with cognitive emotional aspects of women. women worried about their physical appearance likely experience sexual dysfunction, since their sexual excitement depends on their sense of being charming to a large extent. on the other hand, in terms of sexual function and marital adjustment, the most important factor for women is not only their ability for sexual activity but also the feeling and belief about body image and the status of their femininity. in a healthy marriage, the existence of a desirable sexual relationship to provide satisfaction to the partners has a fundamental important role in the success and stability of the family. appropriateness and balance in the male and female sexual desires are among the most important causes of happiness and success of married life. disregarding the sexual instinct in humans in most cases causes disturbance in marital relations and it has been observed that the main reason for 80% of marital discords has been sexual dissatisfaction of husbands and wives. on the other hand, when a couple realizes about their infertility, their body images of themselves may be corrupted and their sexual activities may be damaged. infertility evaluation and using assisted reproductive techniques may have negative effects on the body image of the female and her emotions about her sexual value. some researchers believe that body image in fertile women is partly associated with sexual function. in the study of pujols et al., it was demonstrated that by having better body image in women, their sexual functioning and satisfaction had been more desirable. in the study of meltzer that aimed to determine body image, marital satisfaction, and the sexual satisfaction in couples , it was found that whenever the body image was stronger, marital and sexual satisfaction was better. the study of kilic et al. in patients with permanent ostomy indicated that in these patients, better body image is triggered stronger sexual relationship and greater marital adjustment. in contrast, knafo et al. conducted a study on checking the relationship of body image satisfaction and sexual performance in women with disseminated sclerosis disease. the obtained showed that distress and concern regarding the body image were not associated with reduced sexual functioning. considering the of various studies about the influence of body image on sexual consequences as well as increased rate of problems caused by sexual and marital relationship issues, and also the lack of a study to investigate body image and its relationship with sexual function and marital adjustment in infertile women, it seemed that conducting a study in this field is necessary. therefore, the researchers decided to study the body image in infertile women and its relationship with their sexual function and marital adjustment. we hope that using the of this study, policy makers could provide suitable treatment guidelines as well as appropriate counseling and educational programs to alleviate the sexual and marital problems of infertile women and also to improve their mental health. this correlational study was carried out on 130 infertile women who referred to montaserieh research treatment infertility centre in mashhad, iran in 2011. the sample size calculation was carried out by conducting a pilot study on 20 eligible infertile women using correlation formula based on the correlation coefficient between body image and marital adjustment using 95% confidence interval and a power of 80%, and it was estimated that a sample size of 130 was needed for each group. the participants who met the following criteria were included in the study: being able to talk in persian, having the ability to read and write, being in the age range of 2040 years, not being pregnant after a minimum of 1 year of unprotected sex, and having primary infertility confirmed by a gynecologist. the exclusion criteria were: having undergone training or counseling sessions about sexual issues or marital relationships in the last month, having a history of medical illnesses or any physical, mental, and movement disabilities, being under treatment due to sexual dysfunction (lack or loss of libido, sexual aversion, lack of sexual pleasure, fearing of having sex, sexual motivation disorder, vaginal dryness, orgasmic dysfunction, painful intercourse, vaginismus, or extreme sexual stimulation), taking medications to increase or decrease libido (libido - enhancing drugs including ephedrine, caffeine, synarlyn, levothyroxine, danazol, herbal medicines like aphrodite and rose or damask rose fragrances, and libido - lowering medications such as spironolactone, hydrochlorothiazide, clofibrate, digoxin, propranolol, fluoxetin, haloperidol, clonidine, chlordiazepoxide, and methyl dopa), women with a history of aggression or sexual harassment, having undergone surgery in the past 6 months, having a mental illness, and drug use or alcohol consumption. to collect data, valid and reliable questionnaires including demographic and infertility - related data questionnaire, modified younesi body image questionnaire, rosen female sexual function index (fsfi), and dyadic adjustment scale (das) were used. body image assessment questionnaire was developed based on a six - point likert scale by george kelly mental structure method. this questionnaire was validated in 2001 by younesi and colleagues in iran to measure the dimensions of body image. the mentioned questionnaire had 66 questions with six subscales including the body during activity and when alone, the real body, the ideal body, the body as people see and know, the body as it is recognized by the spouse, and the body as it is known by the spouse's family. in the body image assessment questionnaire, each question was rated with a minimum of one and a maximum of six points. the maximum score that could be obtained in the questionnaire was 396 and the minimum score was 66. higher score indicated more positive or better body image and lower score demonstrated more negative body image. this questionnaire was developed in 2000 by rosen and colleagues with 19 questions and six subscales including libido, sexual excitement, vaginal moisture, orgasm, painful intercourse, and sexual satisfaction. the scores given were: 15 points for questions regarding the scope of libido, 05 points for sexual excitement, vaginal moisture, painful intercourse, and orgasm, and zero or 15 points for questions regarding sexual satisfaction. the cut - off score of sexual function scale was 28, and the minimum score for the total scale was considered 2 and the maximum was 36. generally, higher scores indicated better sexual function. it was used to assess four elements of adjustment including marital satisfaction, marriage coherence, agreement in marriage, and manifestation and expression of couples feelings and emotions. this questionnaire had 32 questions with a maximum score of 151 and a minimum score of zero. scores of less than 100 in this scale meant lower marital adjustment and scores above 100 indicated greater marital adjustment. the validity of the modified questionnaire of body image assessment, women's sexual function, and marital adjustment was determined by content validity method. these tools were created by reviewing the most recent books and articles in the field of the study subject. then, they were given to 10 experts for the needed evaluation and finally, the corrected comments were applied in the developed tools. reliability of body image assessment questionnaire was approved by the internal consistency reliability method by calculating the cronbach's alpha coefficient. which was = 0.92, for women's sexual function questionnaire = 0.79, and for marital adjustment questionnaire = 0.92. for data collection, after obtaining the necessary permission from the montaserieh centre officials and after expressing the objectives and methods to the participants, the researcher obtained the consent of subjects and reassured them about confidentiality of the information; they were explained that their participation in the study was completely voluntary and they could withdraw from the study whenever they wanted. they were also assured that the research would be published as a whole and in the format of study groups information, and individual would be supplied if necessary, anonymous demographic characteristics. demographic and infertility information questionnaire and marital adjustment scale were completed through subjects self - report. body image and sexual function questionnaires data description was performed using frequency tables, graphs and mean indicators, median and standard deviation. the followings tests were used for statistical analysis: pearson and spearman correlation coefficients to determine the relationship among the normal and abnormal quantity variables, independent t - test (student 's t - test) for the comparison of means of two - state quality variables, and analysis of variance (anova) and tukey post hoc test for the comparison of means of multi - complex variables (normal quantitative and multimode qualitative variables) to examine the pure relationship between the variables for direct and indirect effects of confounding variables on the main variables. this study has been approved (with the registration number of 89050) by the regional ethics committee of mashhad university of medical sciences, mashhad, iran. ninety - eight women (75.4%) were housewives. in terms of job categories of infertile women, 19 subjects (59.4%) were involved in cultural educational fields and other occupational categories such as medical sciences, technical, and engineering. thirty - eight women (29.2%) had university degree, 43 subjects (33.1%) had high school education, and the others had primary and middle school education. forty - seven (36.2%) infertile women's spouses were self - employed, 40 (30.8%) spouses were employees, and others were either workers or unemployed. one hundred and six (81.5%) infertile women lived only with their respective partner in one place and 106 (79.2%) subjects lived in the town. in terms of housing status, 53 (40.8%) women had a private home and 49 (37.7%) had a rental property. of the study showed that 99 (76.2%) infertile women had social and artistic outdoor activities (other than their permanent jobs). thirty - five (54.7%) subjects attempted to increase or decrease their weight to remain fit. the mean of body mass index (bmi) of the women was 20.87 3.71. other of the study showed that the mean length of marriage was 7 4.23 years. the mean period of knowing about their infertility was 5.24 4.12 years and the mean duration of the infertility treatment was 4.1 3.95 years. the cause of infertility in 61 cases (46.9%) was female factor, in 49 cases (37.7%) was male factor, and in the remaining was a combination of male and female factors or unknown. forty - nine cases (38.0%) were affected by sperm disorders and 43 cases (33.3%) with ovulation disorders. in addition, 74 (56.9%) infertile women were quite promising about the success of their treatment and 100 (76.9%) women were accompanied by their spouses in the process of infertility treatment. one hundred and sixteen (89.2%) of the women themselves and 115 (88.5%) of their spouses had a great desire to have children. the mean sexual function score was 27.23 3.8 and the mean score of marital adjustment was 113.8 19.73. in terms of body image, 121 (93.1%) infertile women had higher scores than the mean score. fifty - nine (45.4%) infertile women had sexual function scores more than the cut - off point of 28 on the scale of women's sexual functioning. one hundred (76.9%) women had high marital adjustment score (above the cut - off score of 100). the of pearson correlation test showed that there was a direct correlation between overall body image of infertile women with sexual function (p < 0.001, r = 0.4). spearman correlation test showed that there was a direct correlation between the subscales of ideal body image (or desired) with overall sexual function (p = 0.04, r = 0.2). pearson test showed a direct correlation between the subscales of body image, including the body when alone (p < 0.001, r = 0.305), the real body (p = 0.008, r = 0.231), the body that people think (p < 0.001, r = 0.330), the body as it is recognized by the spouse (p < 0.001, r = 0.4), and the body as it is known by the spouse's family (p < 0.001, r = 0.360), with sexual function. relationship between body image subscales with sexual function in infertile women in addition, spearman correlation test showed that there was a direct correlation between overall body image with marital adjustment in infertile women (p < 0.001, r = 0.4). in addition, spearman test showed that there was a direct correlation between body image subscales, including the body when alone (p = 0.001, r = 0.312), the real body (p = 0.004, r = 0.353), the body that people think (p = 0.016, r = 0.211), spouse's imagination of the body (p < 0.001, r = 0.453), and spouse's family's imagination of the body (p < 0.001, r = 0.402), with marital adjustment in infertile women. therefore, by improving the body image subscales in infertile women however, there was no relationship between the subscales of ideal body image with marital adjustment. relationship between body image subscales with marital adjustment in infertile women pearson's test on the correlation between body image with sexual function subscales in infertile women showed that there was a direct correlation between body image with sexual arousal subscale (p = 0.003, r = 0.3). according to the spearman test , there was a direct correlation between the body image and subscales of sexual desire or attraction (p = 0.024, r = 0.2), vaginal moisture (p = 0.001, r = 0.215), orgasm (p < 0.001, r = 0.37), sexual satisfaction (p < 0.001, r = 0.34), and dyspareunia (p = 0.007, r = 0.235). relationship between body image with sexual function subscales in infertile women in this research, the correlation between body image with marital subscales in infertile women was also evaluated. the of pearson test showed that there was a direct correlation between the body image with the subscales of marital adjustment in terms of agreement and consent (p < 0.001, r = 0.335). spearman's test showed that there was a direct correlation between body image of infertile women with the subscales of satisfaction with life (p < 0.001, r = 0.315), continuity of life (p = 0.007, r = 0.237), and expressing emotions within the family environment (p < 0.001, r = 0.37). thus, with more positive body image in infertile women, the subscales of marital adjustment were found to be better. the of the present study showed that there was a direct correlation between body image with sexual function. there was a direct correlation between the subscales of body image, ideal body (or desired), the body when alone, the real body, the body that the people think, spouse's imagination of the body, and spouse's family's imagination of the body, and sexual function. several studies have confirmed the relationship between the body image with sexual function in different fields. it could be compared to similar studies such as the study of pujols et al. that reviewed the relationship between sexual function, sexual satisfaction, and body image in healthy women, the study of seal et al. on the relationship between body esteem and sexual function among healthy female students, and the study of hopkins investigating self - esteem, body image, and sexual awareness in 3050 year old unmarried women. these studies investigated the role of body image in sexual function of healthy women. in some other studies for instance, da silva et al. studied the effect of colorectal surgery on sexual function, body image, self - efficacy, and public health in women. fobair et al. investigated body image and sexual problems in young women with breast cancer. also, kilic et al. evaluated the effect of permanent ostomy on body image, self - esteem, marital adjustment, and sexual function and jun et al. compared the impact of structured programs on the marital relationship, body image, and sexual function in women with breast cancer and healthy women. the of our study were congruent with the findings of the above - mentioned studies. we argue that in the studies of da silva et al., fobair et al., kilic et al., and jun et al. , women suffered from various diseases which affected their body image; but in our study, although the infertile women were apparently considered as healthy people, they might have the same mental emotional problems which could impact on their body image like other patients. in addition, from the perspective of infertile women, due to impaired fertility, they may assume themselves as sick and think that there is a disorder in their body and, therefore, their body image is affected. knafo et al. aimed at investigating the relationship of body image satisfaction with sexual function in 117 women with systemic sclerosis in their study and indicated that concerns about body image was not associated with reduced sexual function. lack of consistency between our and the findings of knafo et al. was probably because the women who suffered from systemic sclerosis due to their physical deformities were not satisfied with their bodies and, therefore, had no positive body image. researchers believe that people with disabilities and physical deformities feel ashamed of their bodies and are affected by disorders in body image. it is noteworthy that the beliefs of individuals about the deficits and physical deformities have two modes, i.e. either the persons are affected by the defect and it somehow in adverse consequences in their life or they do not pay attention to body defects and problems and assume that there is no defect in their body, and therefore, the defect does not have a negative impact on their personal and social life. the obtained from the present study showed that with more positive body image, there was more favorable sexual function, while in the study of knafo et al., despite the presence of physical defects and negative body image in women due to emotional psychological problems from the disease, they had more emotional feelings to their husbands and despite low body image, no change occurred in their sexual relationships. in addition, the of the present study showed that there was a direct correlation between body image and marital adjustment, i.e. more positive body image was associated with higher score of marital adjustment. also, there was direct relationship between the subscales of body image, including the body when alone, the real body, the body that people think, spouse's imagination of the body, and spouse's family's imagination of the body, and marital adjustment. however, there was no relationship between marital adjustment and the subscale of ideal body. meltzer and mcnulty, in a study to determine body image, marital satisfaction, and sexual satisfaction in women, and also kilic et al. and jun et al. in their studies concluded that more positive body image leads to better marital adjustment and intimacy. the of all three studies were consistent with the of the present study. in a study on the adult urban chinese women, concluded that women who had no good body image did not have a good relationship with their spouses. showed in that study that the women who felt that their husbands had a pretty good idea about their bodies had a positive impression of their own bodies in addition to having better marital relationships. in the study of markey et al. on the role of spouses in the perception of body image satisfaction, it was reported that more satisfaction of women about their bodies had been caused when they felt that their spouses were also satisfied with their bodies and, therefore, this issue ed in better marital relationships. this finding was consistent with the of the present study about direct relationship between the thought of the spouse about the body with marital adjustment in women. during the study, some variables could not be controlled by the researcher; so, they are considered as limitations. for example, stresses and concerns related to counseling, diagnosis, and treatment of infertility in the specialized infertility clinic of montaserieh might have an impact upon the subjects regarding how they respond to the questions. in this context, it has been tried to complete the questionnaires at a proper time with necessary mental preparation of the subjects. for proportional control of these restrictions, despite assuring subjects about the confidentiality of their information, in some cases they could not respond to the questions correctly. also, personality differences of the subjects could affect the way they answer to the questions, which could not be controlled by the researcher. therefore, it is suggested that the relationship between body image with sexual function and marital adjustment in different cultures of urban and rural communities should be explored in future research. also, it is suggested to compare the relationship between body image with sexual function and marital adjustment in the general population and the infertile couples population who are undergoing ivf (in vitro fertilization), iui (intrauterine insemination), and other arts (assisted reproductive technology). also, comparing the body image of infertile couples with male and female factors and its impact on their sexual function and marital adjustment is also recommended for further research. in the present study, a direct correlation was found between the body image with sexual function and marital adjustment. by improving the body image in infertile women, a better sexual function and higher marital adjustment the of this study can draw the attention of policy makers to develop and suggest training programs for the health professionals working in infertility clinics, specialized clinics for women, and psychology and family counseling clinics. also, it is recommended to assess body image, sexual function, and marital adjustment of infertile couples when they are admitted to any treatment or counseling center, in order to offer them appropriate training program to develop their positive body image, which in turn in better sexual function and marital adjustment. the of this research could also contribute to the level of awareness and knowledge of teachers, students, and health care providers, who either work in general health centers or special infertility research centers, regarding the mental health of infertile women.

: body image is related to cognitive, emotional, and physical aspects of women's life. therefore, it is expected to have an important role in women's sexual health and marital adjustment too. this issue seems to be salient in infertile women who suffer from psychological consequences of infertility. this study was conducted to investigate the relationship of body image with sexual function and marital adjustment in infertile women in 2011 in mashhad, iran.materials and methods: this correlational study was performed on 130 infertile women who referred to montaserieh infertility research centre in mashhad, iran. subjects were selected using convenient sampling method. to collect data, valid and reliable questionnaires including demographic and infertility - related data tool, modified younesi body image questionnaire, rosen female sexual function index (fsfi), and spanier dyadic adjustment scale (das) were used. data analysis was performed by spss software using student's t - test, correlation, analysis of variance (anova), and tukey post - hoc test.:the mean scores of body image, sexual function, and marital adjustment in women were 308.1 45.8, 27.23 3.80, and 113.8 19.73, respectively. there was a direct correlation between overall body image and subscales of sexual function including sexual arousal (p = 0.003), sexual desire (p = 0.024), vaginal moisture (p = 0.001), orgasm (p < 0.001), sexual satisfaction (p < 0.001), and dyspareunia (p = 0.007). a direct correlation was also observed between overall body image and subscales of marital adjustment including agreement and consent (p < 0.001), satisfaction with life (p < 0.001), continuity of life (p = 0.007), and expressing emotions within the family environment (p < 0.001).: improved sexual function and marital adjustment in cases with higher body image provides evidence that one of the solutions to reduce sexual dysfunction and marital dispute in infertile women could be planning educational and counseling programs to improve women's body image.

a nonhealing corneal ulcer is defined as an ulcer which does not show any indication of healing within two weeks, despite the administration of proper medical treatment. suspicious causes of a nonhealing corneal ulcer include persistent infection, neurotrophic keratopathy, exposure keratopathy, dry eye, treatment toxicity, steroid use, and chronic conjunctival inflammation, such as ocular cicatricial pemphigoid. once a corneal ulcer occurs and is left unattended, corneal melting, descemetocele, and corneal perforation can subsequently develop, leading to devastating consequences. moreover, when a resistant corneal ulcer progressively develops, lamellar keratoplasty or penetrating keratoplasty is usually needed. therefore, curing a resistant ulcer in its early stages is highly recommended. amniotic membrane transplantation (amt) can be used to treat a superficial corneal ulcer. however, the efficacy of amt, or even repeated amt, in treating a nonhealing corneal ulcer is limited because the latter is usually associated with severe inflammatory response, which frequently causes corneal melting, earlier amniotic membrane dissolution, and deferred ocular surface epithelialization. thus, reducing inflammatory cell infiltration and improving the local microenvironment may facilitate the healing of a resistant corneal ulcer. inspired by the significant anti - inflammation effects of the epithelial cells and the successful clinical application of laser - assisted subepithelial keratectomy (lasek), which makes an epithelial flap with alcohol and significantly prevents haze formation in the subepithelial area, we attempted to treat nonhealing corneal ulcers in this study with the use of active pedicle epithelial flap transposition combined with amt. this study was approved by the institutional review board of shandong eye institute, qingdao, china, and conformed to the guidelines of the declaration of helsinki. the medical records of patients who had undergone active pedicle epithelial flap transposition combined with amt for a nonhealing corneal ulcer between 1 march 2012 and 1 july 2015 were reviewed. all of the patients had a history of eye hyperemia associated with pain and decreased vision. the ulcers were from 2 to 5 mm in diameter and less than 1/3 of the corneal stroma in depth. all ulcers were associated with corneal stroma melting and remained unhealed for 4 weeks despite the administration of medical treatment. the study mainly focused on nonhealing sterile corneal ulcers, and all patients received corneal scraping, culture, and laser scanning confocal microscopy examination to exclude active infection, for example, a fungal, resistant bacterial, or acanthamoeba corneal ulcer. the amniotic membrane (am) was prepared and preserved as previously reported, with minor modifications. necrotic corneal tissue was removed from the base and wall of the ulceration, and then thermal cautery was applied to the ulcer to dry out its surface. thereafter, an epithelial flap was constructed from the transparent cornea at the edge of the ulcer, near the limbus. for this procedure, obtaining a flap from the edge of ulcer ensures easy transposition, and taking a flap from the cornea near the limbus guarantees rapid healing of the epithelial defect ing from making a flap. in addition, a corneal epithelial scraper was used to create an epithelial flap without the use of alcohol to maintain epithelial cells activity. briefly, the epithelial scraper was gently pressed onto the corneal surface to make a boundary of the flap shaped like the ulcer under the auxiliary arm of a caliper. a pedicle, like a sprout, was preserved in the lateral or nasal portion of the epithelial flap. the corneal epithelium was then carved following the boundary, with the depth confined to the epithelial layer. in this way, next, the flap was rotated to cover the epithelial nonhealing region of the ulcer. to maintain flap adhesion, fluid on the ocular surface finally, a trephine was used to make an amniotic membrane of 14 mm in diameter, and the am patch was sutured onto the surface to cover the entire cornea using a continuous 10 - 0 nylon suture within 1 mm of the limbus (figure 1). the primary diseases of nonhealing corneal ulcer in the study included herpes simplex keratitis (hsk), corneal graft ulcer, and stevens - johnson syndrome (sjs). postoperatively, tobramycin and dexamethasone eye drops (alcon, puurs, belgium) were used 4 times daily for 1 - 2 weeks and then replaced with 0.1% fluorometholone eye drops (santen, osaka, japan) 4 times daily for approximately 1 - 2 months. for eyes with hsk, antiviral medication was applied topically and systemically with adjuvant corticosteroid eye drops. acyclovir eye drops (wuhan wujing pharmaceutical co., wuhan, china) were used 4 times every day. ganciclovir ophthalmic gel (hubei keyi pharmaceutical co., wuhan, china) was used every night. acyclovir was administered orally (8 mg / kg) 3 times daily for 3 months. for eyes with prior corneal transplantation or stevens - johnson syndrome, 1% cyclosporine eye drops (huabei pharmaceutical co., shijiazhuang, china) were used 14 times daily. the medical treatment was adjusted with the alleviation of symptoms and the dissolution of am. corneal fluorescein staining was performed daily and the sutures were removed 13 weeks postoperatively when the am dissolved. patients were examined daily for the first week, weekly for 4 weeks, and monthly thereafter. ulcer healing time was observed by corneal fluorescein staining during the follow - up. the uncorrected visual acuity (ucva), intraocular pressure (iop), and corneal status were recorded. laser scanning confocal microscopy was applied to determine the extent of local inflammation, and anterior segment optical coherence tomography (as - oct) was performed to visualize corneal tissue cicatrization proximal to the ulcer. ucva before and after surgery was converted to the minimum angle of resolution (log mar) for calculation purposes and compared with student's t - test eleven patients (11 eyes) with a nonhealing corneal ulcer who underwent the combined surgery were included in the study. of these, 7 were male and 4 were female, with an age range of 2968 years (mean standard deviation, 58.1 11.4). the etiologies of the corneal ulcers included hsk (8 eyes), corneal graft ulcer (2 eyes), and sjs (1 eye). the ulcers were less than 1/3 of the corneal stroma in depth and 25 mm in diameter and lasted for 16 months. the site of ulcer in the 11 cases was on the pupillary zone in 5 eyes, on the inferior cornea in 5 eyes, and at the superior cornea in one eye. these patients underwent various medical treatments (antibiotics, antiviral medication, corticosteroids, growth factor, and bandage contact lens) for 4 weeks. of these, 5 eyes received amt once and 1 eye received amt twice, but the ulcers remained unhealed (table 1). thereafter, active pedicle epithelial flap transposition combined with amt was performed, and patients were followed - up for 624 months (16.9 9.9). after surgery, the corneal ulcers healed from 6 to 15 days (10.8 3.1) postoperatively with negative fluorescein staining. the epithelial defect that ed from the construction of flap healed within 1 - 2 days (1.45 0.52). following surgery, corneal opacity was alleviated during the follow - up, with variable effects on visual acuity (va) (figure 2). an elevated iop (30 mmhg) was present in 1 eye and controlled by medication within 3 days. hsk recurrence was observed in 1 patient at 10 weeks and 14 months but without the occurrence of corneal ulcer. corneal ulcer recurrence was noted in 1 eye (after penetrating keratoplasty) at 3 weeks postoperatively, ing in a fungal infection and intractable corneal perforation. the average preoperative and postoperative ucva were 1.70 log mar and 0.82 log mar, respectively. the percentage of patients with ucva of < 0.05 decreased from 73% (8/11 eyes) preoperatively to 9% (1/11 eyes) postoperatively. the va increased by at least one row (maximum, six rows) after surgery. the difference in ucva pre- and postoperatively was statistically significant (p = 0.001) (table 2). the inflammatory cells were found to aggregate in the epithelium and basal membrane surrounding the epithelial nonhealing region of the ulcer by confocal microscopy prior to surgery. inflammatory cell infiltration was noted to be greatly alleviated after surgery (figure 3). as - oct revealed that the surgical treatment had healed the corneal ulcer. postoperatively, corneal stromal edema decreased, leaving a semitransparent and highly reflective region in the cornea (figure 4). the management of nonhealing corneal ulcers is one of the most difficult challenges faced by ophthalmologists because only a few resistant ulcers can be cured solely by medication. once the nonhealing corneal ulcer tends toward expansion and aggravation, devastating complications can subsequently develop. in the current study, the combination of active pedicle epithelial flap transposition and amt was found to be effective in promoting ulcer healing and yielding a good cosmesis. following the treatment, complete ocular surface epithelialization was achieved within an average of 10.8 days. initially understanding the underlying reasons for a deferred epithelial healing in this case series was necessary to achieve the most optimal therapeutic effect and recognize the involved mechanisms of the combined surgery in treating resistant corneal ulcers. firstly, an abnormal or deficient basal membrane caused by corneal melting due to local inflammation hindered epithelial healing, ing in defective cellular adhesion and recurrent breakdown of the epithelium. secondly, the inflammatory cells and stromal keratocytes in a distinct, preexisting inflammatory microenvironment restrained epithelial healing by secreting proinflammatory cytokines and proteolytic enzymes. in this study, the primary diseases of corneal ulcer were hsk, corneal graft ulcer, and sjs. it is possible that many of the eyes affected by hsk or after keratoplasty had poor corneal sensation and the eyes with sjs had limbal stem cell pathology. we believe that the mechanism behind the combined surgery in promoting ulcer healing is related to a comprehensive improvement in the corneal microenvironment through the use of an active epithelial flap. since the introduction of lasek to refractive surgery in recent decades, epithelial flaps have successfully been used in subepithelial keratectomy by covering the excimer laser ablation area to reduce inflammation and scarring caused by photorefractive keratectomy. it was shown in previous studies that many mediators produced or expressed in the corneal epithelium were effective in regulating inflammatory response and maintaining the homeostasis of the ocular surface, such as resolvin d1, resolvin e1, il1ra, netrin-1, and unc5b. these factors reduced the recruitment of inflammatory cells, enhanced phagocytosis, and suppressed the secretion of proinflammatory cytokines. it has been demonstrated in previous researches that active epithelial cells can markedly reduce ocular surface inflammation and relieve neurotrophic keratopathy. the flap was obtained without the use of alcohol in the current study to preserve the activity of the epithelial flap as far as possible. the active epithelial flap inhibited inflammatory cell infiltration in the inflamed tissue and reduced the quantity of proteinases and cytokines released into the inflammatory cornea. covering the ulcer with the active epithelial flap provided a relatively healthy substrate and microenvironment. this facilitated epithelial migration, reinforced basal epithelial adhesion, and promoted ocular surface healing. meanwhile, a decrease in the number of inflammatory cells was detected on confocal microscopy. in addition, thermal cautery was helpful in astringing the melting tissue, gaining better adhesion of the epithelial flap, and ensuring a relative healthy basal membrane. debridement of the necrotizing tissue around and on the ulcer base helped to improve the corneal microenvironment for ulcer healing. by contrast, a replicating virus and/or severe local inflammatory response could damage the corneal basal membrane and disturb the normal epithelium repair process. following surgery , the topical administration of corticosteroids further suppresses the immune response, alleviating the stromal edema and improving the visual acuity. of all the eyes, 5 eyes received amt once and 1 eye received amt twice. after the application of a combination of active pedicle epithelial flap transposition and amt, all of these ulcers were successfully cured within a short time. the indicate that the epithelial flap might have a greater anti - inflammatory and prohealing effect than that of the amniotic membrane. we speculated that it was the collaborative effect of the epithelial flap and amniotic membrane which inhibited inflammatory cell infiltration and facilitated a relatively healthy microenvironment for ulcer healing. a high risk of immune rejection, epithelial defect, infection, graft melting, and corneal perforation is usually encountered when treating ulcers by corneal transplantation in patients with sjs or other immune diseases. there is less possibility of the immune system being activated and more opportunity of gaining a favorable va prognosis by employing autogenic epithelial flap transposition in combination with amt, in addition to reconstructing the corneal surface at an early stage. in the current study, it was a considerable concern of the clinician whether or not the construction of an epithelial flap would aggravate the primary ocular disease. the epithelial flap was obtained from the transparent cornea close to the limbus for all the eyes because the limbal stem cells are located in this position and taking a flap from here would assure rapid epithelialization. the epithelial defect that ed from making the flap healed 1.45 days postoperatively and no new region of epithelial defect was identified. thus, active pedicle epithelial flap transposition combined with amt was a simple, safe, and effective treatment for nonhealing corneal ulcers. the treatment of nonhealing sterile corneal ulcers was addressed in the current study but the same approach could not be expected to be effective in patients with recalcitrant corneal ulcers associated with active fungal or bacterial infection. it is unknown whether the method should be delayed until all conventional treatment has failed or instead be considered earlier. although the outcome of combined surgery was good in the current study, a randomized, controlled study with a larger sample size is needed for further investigations. the combination of active pedicle epithelial flap transposition and amt can reduce inflammatory response, promote epithelial healing, and restore useful vision in cases of nonhealing corneal ulcers.

introduction. the objective was to evaluate the efficacy of active pedicle epithelial flap transposition combined with amniotic membrane transplantation (amt) in treating nonhealing corneal ulcers. material and methods. eleven patients (11 eyes) with nonhealing corneal ulcer who underwent the combined surgery were included. postoperatively, ulcer healing time was detected by corneal fluorescein staining. visual acuity, intraocular pressure, surgical complications, and recurrence were recorded. corneal status was inspected by the laser scanning confocal microscopy and anterior segment optical coherence tomography (as - oct). . the primary diseases were herpes simplex keratitis (8 eyes), corneal graft ulcer (2 eyes), and stevens - johnson syndrome (1 eye). all epithelial flaps were intact following surgery, without shedding or displacement. mean ulcer healing time was 10.8 3.1 days, with a healing rate of 91%. vision significantly improved from 1.70 to 0.82 log mar (p = 0.001). a significant decrease in inflammatory cell infiltration and corneal stromal edema was revealed 2 months postoperatively by confocal microscopy and as - oct. corneal ulcer recurred in 1 eye. none of the patients developed major complications. . active pedicle epithelial flap transposition combined with amt is a simple and effective treatment for nonhealing corneal ulcers.

we review 4 patients with severe gbs and one patient with miller fisher syndrome (mfs) admitted to our institution from 2012 to 2013. all patients received very high - dose ivig (total dose of at least 4 g / kg, equivalent to 2 standard treatment courses) administered over fewer than 14 days. all patients with gbs did not respond initially, prompting a second course of treatment (table). as this was a retrospective review of a small case series, institutional review board approval was not required. all of the patients with non - o blood types (3/5) treated with very high - dose ivig developed clinically significant anemia requiring blood transfusion. when tested (2/3 cases), there was clear evidence of hemolysis (positive direct antiglobulin test, low haptoglobin, high lactate dehydrogenase / bilirubin). in addition, there were no other medical comorbidities or medications commonly implicated in causing autoimmune hemolytic anemia (table). patient 4 developed hematuria prior to discharge with normal hemoglobin and returned with massive hemolysis leading to pigmentary nephropathy and requiring hemodialysis. patients with o blood type did develop anemia, but it was mild and clinically asymptomatic. in addition, the patients with o blood type did not have evidence of hemolysis. antibodies to blood group antigens (a and b) found in ivig due to pooling from donors with o type blood are proposed as the hemolytic mechanism in many prior observations. the current industry standard antibody titers are < 1:64 for anti - a and < 1:32 for anti - b. privigen, a commonly used liquid ivig formulation, currently has anti - a < 1:32 and anti - b < 1:16. however, the titers of anti - a and anti - b may vary within lots. therefore, patients who are given higher doses of ivig will be exposed to higher levels of anti - a and anti - b antibodies. although our patients received privigen, ha has been reported with use of many liquid ivig formulations. antibodies to blood group antigens (a and b) found in ivig due to pooling from donors with o type blood are proposed as the hemolytic mechanism in many prior observations. the current industry standard antibody titers are < 1:64 for anti - a and < 1:32 for anti - b. privigen, a commonly used liquid ivig formulation, currently has anti - a < 1:32 and anti - b < 1:16. however, the titers of anti - a and anti - b may vary within lots. therefore, patients who are given higher doses of ivig will be exposed to higher levels of anti - a and anti - b antibodies. although our patients received privigen, ha has been reported with use of many liquid ivig formulations. we propose that risk of ivig - related ha is increased with high doses, significant in non - o blood types, and more likely to occur when administered over a short interval (i.e., < 2 weeks). ha has not been frequently observed in patients receiving maintenance ivig, likely due to the lower dose, longer time intervals between infusions (typically 34 weeks), and 23 week half - life of anti - a / b igg. although all of our patients had gbs / mfs, ha has been reported in many other conditions treated with high - dose ivig such as postpolio syndrome, chronic inflammatory demyelinating polyneuropathy, myasthenia gravis, renal transplantation, and kawasaki disease. the applicability of our findings is limited by a small number of cases, retrospective analysis, and hemolysis testing in only 2 of the 3 patients. however, our findings raise relevant, novel considerations such as a possible maximum tolerated dose of ivig in a short time frame. in addition , practitioners should exercise caution if considering escalating ivig dosages to > 2 g / kg in < 14 days. hemoglobin and creatinine levels should be monitored closely during and after treatment with high - dose ivig (up to 12 weeks) to detect this untoward complication. in the literature , nadir hemoglobin level seemed to occur within 2 weeks of treatment, commonly within 45 days. if anemia occurs, testing should include direct antiglobulin test, peripheral blood smear, bilirubin, haptoglobin, lactate dehydrogenase, and reticulocyte count. manufacturers should label anti - a and anti - b titers on their product and consider more stringent industry standards. we propose that there is a maximal tolerated dose of ivig over a 2-week period, which is dependent on patient abo blood group and ivig's anti - a / anti - b titers.

objective: we describe an underrecognized side effect of high - dose iv immunoglobulin (ivig), hemolytic anemia.:there are no established guidelines on treating patients with guillain - barr syndrome (gbs) who relapse or do not improve after a standard course of treatment (ivig or plasma exchange). some centers will opt for a second course of the initial treatment. there is an ongoing trial of a second course of ivig in patients with severe gbs.methods:we retrospectively reviewed 4 patients with severe gbs who received high - dose ivig. one patient inadvertently received a high dose of ivig for miller fisher syndrome. all patients received a total of at least 2 courses of the standard dose of ivig (total > 4 g / kg). we review their clinical course and side effects.:all patients with non - o blood types developed clinically significant hemolytic anemia requiring blood transfusion.:hemolytic anemia may limit doses of ivig for treatment of severe gbs in patients with non - o blood types.

the effective administrators are those who dedicate themselves to success of the organization not showy presentation of their own achievements. while narcissism is a disordered sense of self - admiration, self - confidence was always a necessary psychological need, which has recently changed into a critical economic need. nowadays, not only knowledge is required for the proper leading of an organization, but also qualities such as independence, self assurance, self - dependence, and inventions, all best outlined as self - confidence are mandatory. according to the previous research, the inventive behavior of the manager is most depends on their communication within the professional environment. despite general agreement on the importance of managers for provocation of personal new accomplishments, the literature is less congruent regarding the role of managers and new inventions. these researches actually evaluate the effect of managers using outcome models; that is, discarding outcomes related to invention and concentrating on those behaviors of managers that positively affected the efficacy. in other words, now we need many who bear at least some levels of self - confidence as the matter of economic progression. the vital role of self - confidence is just as evident as the catastrophes ing from its absence. there are six virtues most valuable regarding development of self - confidence, which all a successful organization should actualize: informative life, self - acceptance, responsibility, assertiveness, directed life, and self - integration. if an individual or an organization represents positive attitudes about a behavior and believes in it, that behavior brings about positive outcomes. if an organization desires to achieve a prominent behavioral culture and brilliant self - confidence, it should consider at least seven basic principles: respect, responsibility and sources, acknowledgment and reward, relationships, icon presentation, and regeneration. these principles are synergistically related and a greater outcome if they concomitantly occur. self - confidence is a complex made of self - efficacy and self - esteem. major life challenges include the ability to balance fiscal affairs, self - care, competent social interactions, and capabilities to overcome obstacles and persistently accomplish their visions. a productive self - confidence contains all these elements and its absence in adverse effects on the physical and emotional state of a human being. those with low self - confidence usually judge themselves based on the current accidents and their negative sensations about themselves persist more than their positive perceptions do. a healthy self - confidence emanates from the ability of true evaluation of oneself. today, educational authorities, parents, financial and governmental leaders, all acknowledge the value of self - confidence in gaining success. self - confidence is related to personality characteristics, capabilities, weaknesses, and other features of psychological order of the person. self - confidence, in fact, is a reflection of foreign evaluations. reviewing literature shows that self - confidence is a psychological concept formed during growth of an individual and may not be expeditiously changed. authorities believe that both the inventive mind and self - confidence belong to acquired characteristics of a human being. in fact, selfishness or more specifically narcissism is the preference of self to others. while selfishness is a common trait, narcissism bears a psychodynamic nature. according to freund s analysis of this personality, disorder the confidence of the sufferer is limited only to the positive points to only accept positive points and reject weaknesses. they believe they are absolute and ideal without defects. the aim of this study was to evaluate the relation between the self - confidence of nursing administrators and vocational satisfaction of the staff in the teaching hospitals of tums, tehran, iran. this cross sectional study was a descriptive analytical study and performed over 6 months from may to november 2011. we collected the detailed list of 16 teaching hospitals under administration of tehran university of medical sciences early 2010 and gathered data via stratified randomized sampling scheme. as there are only a few iranian studies on self - confidence of nursing administration and its correlation to vocational satisfaction of the staff, we arbitrarily choose the ratio of nursing administration self - confidence to vocational satisfaction of staff to be 50% and using the below formula for sample size calculation, n = z1 /22p(1-p)d2,where d was the accuracy (7%) and for the =.05 we computed sample size as 196 and interviewed 200 individuals in each group of nursing administrators and staff. the instrument for measurement was a questionnaire designed by researcher that containing demographic section and nursing administration self - confidence (56 items) and vocational satisfaction (21 items) sections using likert s 5-point scale. however, before the start of the study the questionnaires were considered by colleagues and their comment were implemented to improve the quality of the questionnaires and the content validity found reasonable by them. then, after collecting the data from 30 nursing administrators and 30 staff, the cranach s alpha was calculated on the corresponding questions and found to be 0.95 and 0.97 respectively, which are evidences of high reliability. after the data were collected, we used spss (17.0) for data entry and primary evaluation. we scored the answers for both nursing administrators and staff and divided them into three groups as: 1 ) below 33%, 2 ) 33% to 67% and 3 ) above 68%. the demographic characteristics of the nursing administrators and staff described as a table. also, the ratio of score of self - confidence of nursing administrators and vocational satisfaction of staff in the above three groups were computed and presented as a table. after description of demographic features ratio of scores, the univariate analysis of the effects of personal factors on the self - confidence of nursing administration and vocational satisfaction of the staff was carried out. as the scores were grouped and were ranks we used non - parametric tests such as mann - whitney and kruskal - wallis. in addition, we used spearman s correlation of coefficient. in order to adjust for the effect of dependency of scores in the same department the research ethics was approved by deputy of research of school of public health of tehran university of medical sciences. we described the goal and nature of this study for nursing administrators as consent their questionnaires. the age of 39.5% of administrators was between 3039 years and 9.5% were more than 50 years. most of them were academically trained and had the bsc (47%) or msc (49.5%) degrees. in the staff group, the age of 38.5% of the staff was between 3039 years and 10% were over 50 years. 44.5% of them had academic degree as bsc and 8% had technician degree (table 1). as the table 2 presents, the ratio of administrators with low self - confidence score was 32.5%, moderate score was 34.5% and high score was 33%. furthermore, this table demonstrates that the satisfaction in the staff group was low in 35.5%, moderate in 27%, and high in 37.5%. the mean sd of score value for self - confidence of managers and satisfactory of staff were 134.9 19.8 and 89.12 18.3, respectively. mean sd for self - confidence scores in men and women administrators were 140.3 20.1 and 132.63 19.4, respectively (p=0.016). however, there was no significant difference between self - confidence score of administrators with different age (p=0.69), educational level (p=0.11), and organizational ranking (p=0.35). thus, the confer that the only effective factor in the self - confidence of administrators is their gender. similarly, the satisfaction score was only related to the years of experience of staff (p=0.03) in which satisfaction was lower among those with less than 10 years of experience. in addition, there was insignificant relationship between satisfaction score of the staff and their sex (p=0.19), age (p=0.13), educational level (p=0.28), marital status (p=0.11), number of children (p=0.31), educational level of spouse (0.93), and employment status of spouse (p=0.46). as the distribution of both the satisfaction and self - confidence were not normal (p<0.0001 and p<0.0004 respectively), therefore spearman correlation of coefficient was computed to analyze the correlation between these two variables. preliminary analysis demonstrated a positive correlation between the two so as the higher the self - confidence the higher the staff satisfaction is (p=0.037). then, it may be asked if this correlation is under influence of factors affecting either vocational satisfaction (such as years of employment) or self - confidence (such as gender). in fact, the staff employment history should significantly correlate with the self - confidence of the administrators and the latter gender should correlate with the staff satisfaction. however, this turned not to be the case for either the former (p=0.82) or the latter (p=0.10). it is worth mentioning that all the analysis was carried out without adjusting for the effect of sampling scheme, department effect, means looking at the possible effect of correlation of the variables as the administrators and staffs were from same department. however, since adding this effect will always attenuate the correlations (increase the p - value) and as none of the univariate analysis was significant; therefore the findings will remain the same. then, only we need to look at the association of the self - confidence of the administrators and staff satisfaction in the presence of department effect. after adjusting for the effect of department in a random effect modeling, the p - value of the correlation between self - confidence and the staff satisfaction from 0.037 increased to 0.055. according to our findings, male administrators have more self - confidence than female administrators do. in addition, we found that the employment experiment of the staff is a significant factor in their vocational satisfaction so that the higher the experience the more the satisfaction. after adjusting for department, the correlation between administration s self - confidence and the staff s vocational satisfaction was found not significant. in our previous study to assess self - confidence over managerial staff in the tehran university of medical sciences the organizational ranking of a manager did not affect self - confidence as is seen in the current study. in the previous study, we found a positive correlation between educational degrees of a manager (more self - confidence with phd degree) which was not repeated in the present study. it is worth to note that there was no correlation between age groups and self - confidence. furthermore, we found that there was a significant correlation between administrators self - confidence and the staff vocational satisfaction. our previous about the correlation of administrators self - confidence and the staff vocational satisfaction are in contrast with the of the present study. this difference mainly from the nature of the administration in the two studies: the previous study was done over the official managers while the current study is done over nursing field administration (supervisor, matron, head nurse). the evidence for the effect of organization on the self - confidence comes from previous studies, who demonstrate that self - confidence is under influence of the type of organization, its size, complexity and context. thus, the observed difference from the different population. we could not find a similar study regarding this variable. however, there are studies illustrating the important role of self - confidence in determination of incentives and work dependent behaviors. many studies demonstrate significant and positive relation between self - confidence and person s efficacy the lee study in 2003 demonstrates the positive relation of the self - confidence and improved efficacy. heck et.al also found a positive relation between the quality of communications between managers and staff and self - confidence of administrators (r=0.67). in 2003 lee showed that administrators self - confidence and the interactions of managers and staff are positively correlated (r=0.67). however, the current study shows that the administrators self - confidence not only adds nothing to improvement of the system situation but also in the reduced staff satisfaction. our study is performed in a medical organization while the referring studies were done over non - medical organizations. acknowledging this, there are studies demonstrating the negative correlation between the size of the organization and the administration s self - confidence. thus, type of the organization, its size, and complexity all affect the self - confidence of both the staff and administrators and may differentially influence their efficiency or satisfaction. the aforementioned considerations call the need for more studies in order to define the role of administrators self - confidence and staff satisfaction in the organizational efficiency of patient satisfaction. in other words, the findings from one organization for instance, the from our study on nursing administrator may not be expanded to non - medical managers or even medical official managers. our research was a new subject, with limited resources. considering the lack of research in this area, the from our study on nursing administrator our study are generated from public hospitals, which are located in tehran; therefore, the generalization of the to the other public hospitals in iran should be done cautiously, even though around 50% of these hospitals are operating in tehran. similar studies in other parts of the country might improve the generalizability of this study. in addition, other cross - sectional the studies are beneficial to monitor the ratio and the correlation between the self - confidence and vocational satisfaction. our demonstrate that gender and years of employment are the only factors affecting self - confidence and vocational satisfaction between the administration and staff respectively. the staff employment history was not significantly correlates with the self - confidence of the administrators and the gender was not correlated with the staff satisfaction. self - confidence in the administration was not significantly correlated with vocational satisfaction of the staff. finally, we should emphasize that self - confidence, affect both the staff and administrators and may differentially influence their efficiency or satisfaction. we recommend evaluating the policies, programs, and the processes that may in improved self - confidence in both the staff and administration. ethical issues (including plagiarism, informed consent, misconduct, data fabrication and/or falsification, double publication and/or submission, redundancy, etc) have been completely observed by the authors.

self - confidence is a glorious feature of an effective administrator. their main goal is the organizational success. therefore, we approached this idea by evaluating the self - confidence of nursing administration in tehran university of medical sciences (tums) teaching hospitals and its relation to vocational satisfaction of the staff.methods:in a cross - sectional study, we interviewed 200 nursing administrators and 200 staff in different departments of the tums teaching hospitals using a standardized questionnaire to assess the self - confidence among nursing administrators and staff satisfaction. data were entered in spss (17.0) and analyzed using this software and stata (11.0) using non - parametric tests and spearman s correlation of coefficient. the significant level was set as p<0.05.: of 200 nursing administrators 58 (29%) were male and 142 (71%) were female. mean sd of the self - confidence score for the nursing administrators was 134.9 19.8. among the staff 68 (34%) were male and 132 (66%) were female. the mean sd of the vocational satisfaction for staff was 89.12 18.3. after considering the effect of departments in a regression model, the correlation between nursing administration s self - confidence and the staff s vocational satisfaction was found not significant (p=0.055).: gender and years of employment were the only factors affecting self - confidence and vocational satisfaction between the nursing administration and staff respectively, which not significantly correlated after adjustment.

malignant transformation may occur in mftthis malignant change denotes loss of heterozygosity in clyd genethe differentiation between bcc and immature te represents a diagnostic challenge malignant transformation may occur in mft this malignant change denotes loss of heterozygosity in clyd gene the differentiation between bcc and immature te represents a diagnostic challenge trichoepithelioma (te) is a benign adnexal neoplasm. lesions are rounded, skin - colored, firm papules or nodules, 2 - 8 mm in diameter. they are located mainly on nasolabial folds, nose, forehead, upper lip, and scalp. multiple familial trichoepithelioma (mft) is an autosomal dominant (ad) disorder beginning in childhood and progresses slowly. a 38-year - old female presented with a 27 years history of asymptomatic, skin colored papules. lesions were firm in consistency, ranging from 5 to 10 mms in diameter and were distributed on her face; around eyes, on the nose, nasolabial folds and upper lip. patient's father had similar lesions but there was no affection of other family members. routine investigations including hemogram, urine analysis, liver and renal function tests were non contributary. first patient presented by skin colored papules on: (a) periorbital region (b) nose, nasolabial folds and upper lip excisional biopsy of a representative lesion was done after taking patient consent. histopathological examination of hematoxylin and eosin (h and e)-stained sections revealed superficial dermal, well circumscribed, non capsulated, symmetrical lesion. this lesion was formed of solid aggregates of uniform basaloid cells with peripheral palisading but lacked epidermal connection, pilar differentiation and retraction artifacts. they were surrounded by a stroma with increased number of fibroblasts. cells were uniform, with large nuclei and scanty cytoplasm and lacked cytological atypia, mitoses or necrosis. aggregations of fibroblasts, representing abortive attempts to form papillary mesenchyme (papillary mesenchymal bodies), were detected, that are characteristic of te. (a) trichoepithelioma showing superficial dermal solid basaloid masses with peripheral palisading and lacked the epidermal connection, epithelial stromal retraction artifact, mitosis or cytologic atypia (b) an intrastromal cleft (arrow) was observed around the solid aggregates that were tightly encircled by fibroblasts (h and e, 200 a and b) (c) aggregations of fibroblasts, representing abortive attempts to form papillary mesenchyme (papillary mesenchymal bodies) (arrow) (d) foreign body granuloma (arrows) adjacent to a basaloid mass (asterisk) (h and e, 400 c and d) based on clinical and histopathological criteria, the diagnosis of immature te was reached. however the presence of solid basaloid aggregates with peripheral palisading may lead to a misdiagnosis of bcc. thus, we performed immunohistochemical (ihc) staining for bcl2, cd10, cd34 and androgen receptor (ar) antibodies. both cd10 and cd34 stained the stromal cells but not the basaloid cells. both tumor cells and stroma did not show any ar immunoreactivity. trichoepithelioma ihc staining profile: (a) the outermost epithelial cells showed positivity for bcl2 (arrows) (b) cd10 stained the stromal cells but not the basaloid cells (arrow) (c) cd34 stained the stromal cells (colored arrows) but not the basaloid cells (asterisk), it also stained blood vessel (internal control) (black arrow) (d) both tumor cells and stroma lacked any androgen receptor immunoreactivity (immunoperoxidase 400 for a, b, c and d) a 65-year - old male, the father of the first patient, was presented similarly by papulo - nodular lesions with the same distribution and morphology described in his daughter but larger in size. one month before reaching us, one of those nodules enlarged markedly to reach a size of 6 cm with repeated bleeding, ulceration and crustation. patient's father presented by similar but larger lesions on (a): periorbital region and nose with ulcerated crusted nodule on the left side of the nose (b) excisional biopsy was taken from a representative papule and incisional biopsy was taken from the enlarging nodule after taking patient consent. for the former biopsy, examination of h and e - stained sections and immunohistochemical - stained sections of the same antibodies revealed the same as the first case. for the latter biopsy, islands of basaloid cells extending from the epidermis to the dermis with peripheral palisading and prominent stromal epithelial retraction artifacts were observed. immunohistochemical staining revealed that, the basaloid cells stained diffusely with bcl2 , cd10 and focally with ar antibodies. basal cell carcinoma ihc staining profile: (a) the basaloid cells stained diffusely with bcl2. (c) cd34 immunostaining was negative in basaloid cells and fibrous stroma but stained the blood vessels (internal control) (arrows). (d) androgen receptor stained the basaloid cells (immunoperoxidase 400 for a, b, c and d) a 38-year - old female presented with a 27 years history of asymptomatic, skin colored papules. lesions were firm in consistency, ranging from 5 to 10 mms in diameter and were distributed on her face; around eyes, on the nose, nasolabial folds and upper lip. patient's father had similar lesions but there was no affection of other family members. routine investigations including hemogram, urine analysis, liver and renal function tests were non contributary. first patient presented by skin colored papules on: (a) periorbital region (b) nose, nasolabial folds and upper lip excisional biopsy of a representative lesion was done after taking patient consent. histopathological examination of hematoxylin and eosin (h and e)-stained sections revealed superficial dermal, well circumscribed, non capsulated, symmetrical lesion. this lesion was formed of solid aggregates of uniform basaloid cells with peripheral palisading but lacked epidermal connection, pilar differentiation and retraction artifacts. they were surrounded by a stroma with increased number of fibroblasts. cells were uniform, with large nuclei and scanty cytoplasm and lacked cytological atypia, mitoses or necrosis. aggregations of fibroblasts, representing abortive attempts to form papillary mesenchyme (papillary mesenchymal bodies), were detected, that are characteristic of te. (a) trichoepithelioma showing superficial dermal solid basaloid masses with peripheral palisading and lacked the epidermal connection, epithelial stromal retraction artifact, mitosis or cytologic atypia (b) an intrastromal cleft (arrow) was observed around the solid aggregates that were tightly encircled by fibroblasts (h and e, 200 a and b) (c) aggregations of fibroblasts, representing abortive attempts to form papillary mesenchyme (papillary mesenchymal bodies) (arrow) (d) foreign body granuloma (arrows) adjacent to a basaloid mass (asterisk) (h and e, 400 c and d) based on clinical and histopathological criteria, the diagnosis of immature te was reached. however the presence of solid basaloid aggregates with peripheral palisading may lead to a misdiagnosis of bcc. thus, we performed immunohistochemical (ihc) staining for bcl2, cd10, cd34 and androgen receptor (ar) antibodies. both cd10 and cd34 stained the stromal cells but not the basaloid cells. both tumor cells and stroma did not show any ar immunoreactivity. trichoepithelioma ihc staining profile: (a) the outermost epithelial cells showed positivity for bcl2 (arrows) (b) cd10 stained the stromal cells but not the basaloid cells (arrow) (c) cd34 stained the stromal cells (colored arrows) but not the basaloid cells (asterisk), it also stained blood vessel (internal control) (black arrow) (d) both tumor cells and stroma lacked any androgen receptor immunoreactivity (immunoperoxidase 400 for a, b, c and d) a 65-year - old male, the father of the first patient, was presented similarly by papulo - nodular lesions with the same distribution and morphology described in his daughter but larger in size. one month before reaching us, one of those nodules enlarged markedly to reach a size of 6 cm with repeated bleeding, ulceration and crustation. patient's father presented by similar but larger lesions on (a): periorbital region and nose with ulcerated crusted nodule on the left side of the nose (b) excisional biopsy was taken from a representative papule and incisional biopsy was taken from the enlarging nodule after taking patient consent. for the former biopsy, examination of h and e - stained sections and immunohistochemical - stained sections of the same antibodies revealed the same as the first case. for the latter biopsy, islands of basaloid cells extending from the epidermis to the dermis with peripheral palisading and prominent stromal epithelial retraction artifacts were observed. immunohistochemical staining revealed that, the basaloid cells stained diffusely with bcl2 , cd10 and focally with ar antibodies. basal cell carcinoma ihc staining profile: (a) the basaloid cells stained diffusely with bcl2. (c) cd34 immunostaining was negative in basaloid cells and fibrous stroma but stained the blood vessels (internal control) (arrows). (d) androgen receptor stained the basaloid cells (immunoperoxidase 400 for a, b, c and d) clinically, this tumor occurs either as a solitary lesion without any familial association or as multiple lesions in mft. lesions develop as firm, non - ulcerated, flesh - colored papules with a propensity for the face and rarely exceed 0.5 cm in size. mft was initially described in 1892 under the names multiple benign cystic epithelioma and epithelioma adenoides cysticum. in most cases, ulceration may occur rarely. the gene associated with the familial type of te links to the short arm of chromosome 9. however, other reports documented defects of a tumor repressor gene on chromosome 16, cyld. to date, 17 mutations of this gene have been described to be associated with mft. males and females receive the gene equally, but because of lessened expressivity and penetrance in men, most patients are women. te contains adenoid network or solid aggregates of basaloid cells, horn cysts, and abortive hair papillae.. however, some lesions show relatively little differentiation towards hair structures with very few or even absent horn cysts. histopathological differences observed between te and bcc te is a benign lesion that may be excised by a small margin of healthy tissue, thereby facilitating surgical repair; however, bcc is a locally malignant tumor treated by excision of the lesion with 3 - 4 mm margins. diagnosis may be assisted in a given case by clinical data, and the presence of hereditary transmission. the distinction between bcc and immature te on histopathologic basis is quite difficult especially in small superficially shaved specimens. bcl-2 expression has been found to be limited to the outermost basaloid cells in tumor nests of te and to be diffuse in bcc. cd34 is considered to be a good candidate because the stroma is positive in te and negative in bcc. more recent studies have shown ar expression in a number of mature epithelial structures and epithelial neoplasms including bcc. in contrast, ar expression was absent in mature hair follicles or the few trichogenic neoplasms studied to date. these findings suggested that ar expression might be a useful adjunct in the histologic differential diagnosis between bcc and te. immunostaining markers that differentiate te and bcc malignant transformation of te to bcc is rare. literature review showed that only 12 cases were reported up till now with the first case in 1959. malignant transformation denotes loss of heterozygosity in clyd gene in the 9p21 and 9q22 chromosomal regions. matt et al., had indicated that there is a common gatekeeper between te and bcc. tan and his associates concluded that bcc arising on top of mft lesions may represent a novel contagious gene syndrome. multiple treatment modalities had been suggested for te, including surgical excision and grafting, chemical peeling and co2 laser. the first case had been treated by co2 laser. regarding the treatment of bcc in the second case, it had been surgically excised but he was not interested in treating te lesions. we presented the thirty fourth case of mft and also the thirteenth case of malignant transformation of te to bcc.the histopathological and immunohistochemical differences between te and bcc were summarized and tabulated.we recommend the administration of the suggested immunohistochemical antibodies to differentiate between bcc and immature te. we presented the thirty fourth case of mft and also the thirteenth case of malignant transformation of te to bcc. we recommend the administration of the suggested immunohistochemical antibodies to differentiate between bcc and immature te.

trichoepithelioma (te) is a benign tumor of follicular origin that presents as small, skin - colored papules predominantly on the face. when more than one family member is affected, the disease is known as multiple familial trichoepithelioma (mft). it is a rare autosomal dominant (ad) skin disease. malignant transformation is very rare. we present a case of mft in a female patient and her father with malignant transformation to basal cell carcinoma (bcc) in the father. we summarized the main histological differential parameters between te and bcc and applied immunophenotyping for both by administration of bcl2, cd34, cd10 and androgen receptor (ar) antibodies.

sertoli - leydig cell tumours (sclts) are rare sex - cord stromal tumors of the ovary. retiform sclt's are rare histological variant of sclt with an average age of presentation being 16 years. to the best of our knowledge the retiform variant of sertoli leydig tumors reported so far are seen in less than 10 years of age. we present a rare case of retiform variant of sclt's presenting in a 42-year - old female. a 42-year - old female presented with painless lump in the lower abdomen for 7 years. largest measured 14 12 cm, smallest measured 3 3 cm. on cutting fleshy, firm tissue was found within the cyst wall. microscopic examination showed ovarian tumor with tubular, cord like pattern lined by bland cuboidal to columnar cells. also seen is retiform pattern at few places and scattered cells with abundant eosinophilic cytoplasm suggestive of leydig cells. leydig cells showed focal positivity for vimentin and negativity for leydig cells. histological diagnosis of retiform variant of sertoli leydig cell tumor of ovary stage 1 (t1n0m0) was made. the patient was followed up to 2 years, she did not have any complaints. gross appearance of sertoli leydig cell tumor sertoli cells arranged in cords, sheets and aggregates (he 100) retiform pattern in serrtoli- leydig tumor (he 100) sertoliform cells with clear cytoplasm (he 400) leydig cells showing eosinophilic cytoplasm (he 100) sertoli cells showing atypia (he 400) cytokeratin positivity in sertoli component of sertoli leydig cell tumor of ovary vimentin positivity in leydig component of sertoli leydig cell tumor of ovary (he 100) however, retiform sertoli - leydig cell tumors present in the average age of 16-year - old. clinically, non - virilised patients present with non - specific symptoms like abdominal mass, pelvic pain. it is seen that 80% of the patients with ovarian slcts and virilising manifestations present with elevated serum levels of testosterone and androstenedione. however, sclts are unilateral and are confined to ovary at the time of diagnosis. computerised tomography (ct), magnetic resonance imaging (mri), and positron imaging tomography (pet) scans can provide us a better visualisation of the extra - ovarian or metastatic spread of the tumor. well - differentiated slct have an average diameter of 5 cm whereas intermediate type and poorly differentiated types have an average diameter of 15 cm. microscopically, they are divided into following categories: well - differentiated or meyer type 1: they constitute 11% of slct.intermediate type or meyer type 2: they constitute 54% of slct.poorly differentiated (sarcomatoid or undifferentiated or meyer type 3]: they constitute 13% of slct.sclt with heterologous elements like skeletal muscle, cartilage.retiform type (15%): they resemble rete of ovary or testes. staging wise t1 means tumor limited to the ovaries, t2 means tumor involves one or both ovaries with pelvic extention, t3 means involvement of one or both ovaries with microscopically confirmed peritoneal metastasis outside the pelvis. intermediate type or meyer type 2: they constitute 54% of slct. poorly differentiated (sarcomatoid or undifferentiated or meyer type 3]: they constitute 13% of slct. staging wise t1 means tumor limited to the ovaries, t2 means tumor involves one or both ovaries with pelvic extention, t3 means involvement of one or both ovaries with microscopically confirmed peritoneal metastasis outside the pelvis. a total abdominal hysterectomy is considered as the treatment of choice in patients with unfavourable findings like rupture, extra - ovarian spread, poorly differentiated neoplasm, heterologous mesenchymal differentiation. post - operative chemotherapy, radiotherapy or a combination of both, may also be considered in the patients with above mentioned poor prognostic factors. similarly the 5 year survival rates for stage 1 tumor is 95% and that for stage iii and stage iv is zero percent.

sertoli - leydig cell tumors are the uncommon sex - cord stromal tumors of the ovary. we report a case of 42-year - old female with retiform variant of sertoli - leydig cell tumour. she presented with the complaint of mass in abdomen for 7 years. ultrasound revealed bilateral ovarian mass suggestive of malignancy. bilateral salpingo - oopherectomy with surgical staging was done. the tumor was diagnosed as stage i retiform variant of sertoli - leydig cell tumor on histopathology and immunohistochemistry.

the term taxonomy is used to describe the classification of various things, so the term drug taxonomy refers to the science of listing and describing drugs, according to various properties, in a manner which allows easy comprehension and understanding of their usage. traditionally, only pharmaceutical properties (e.g., chemical structure and pharmacodynamic and pharmacokinetic characteristics) have been used to separate drugs into various groups. increasingly, however, the end user (i.e., the patient s or community s perspective) is considered when studying pharmacology. in the present work, we provide a balanced, syncretic approach to insulin taxonomy, using both patient - centered and pharmacokinetic aspects, to craft a number - based classification of insulin regimes. this article is based on previously conducted studies and does not involve any new studies of human or animal subjects performed by any of the authors. endocrinology and diabetology textbooks provide comprehensive coverage of various insulin preparations and then utilize these to discuss different insulin regimes. the current american diabetes association (ada)/european association for study of diabetes (easd) 2015 guidelines use the terms basal, basal plus, premixed, split - mix, and intensive to describe insulin regimes. other terms used for regimes involving 3 or more injections per day are multiple and intensified insulin therapy. this drug - centered or pharmaceutical - based terminology served diabetology practitioners adequately in the past; the corresponding taxonomic methodology was able to incorporate the limited insulin preparations available, which included both traditional and modern insulins. this pharmaceutical classification of insulin regimes is not, however, syntaxic with the current emphasis on a patient - centered approach. it must be reemphasized here that it is patient - centeredness which forms the basis for recent advances in drug development and improvements in treatment guidelines. most patients of diabetes do not appreciate the pharmacodynamic or kinetic nuances of insulin preparations. what is more relevant to the person requiring insulin is the number of injections to be taken per day, the timing of administration, and the flexibility with which these timings can be adjusted. based upon these factors, it is important to craft a fresh synopsis of insulin regimes, using the number of injections per day as the framework for systematic study. at the same time , such a classification system must address the nature of insulin preparations, whether basal, premixed, or prandial. modern clinical trials are available which support the use of premixed insulin in once - daily and thrice - daily dosages, as opposed to the traditional twice - daily regime. the basal insulins detemir and glargine often need to be prescribed twice daily in order to achieve adequate glycemic control. innovative regimes utilizing combinations of rapid - acting and premixed / coformulated insulins with varying frequencies of administration have also been documented. while a number - based terminology has already been proposed, it is inadequate to cover the current range of insulin preparations and the large number of regimens that they are used in. with the newer insulin analogues available, a modern, number - based classification is required. table 1 lists the various insulin regimes and preparations as well as the frequency and timing of administration for each. all regimes enumerated in this table are backed by randomized controlled trials, as shown in table 2.table 1insulin preparations that are currently on the market, along with the prescription patterns for themfrequency of injectionname of regimeninsulin preparations usedtiming of administration1 (once a day)basalnph, idet, iglar, i glar u300at bedtime or the same time every daybasalidegat any time of the daypremixedbiasp, lispromixwith major mealcoformulationidegaspwith major mealbasal + glp1raideg + liraglutideiglar + lixisenatideat any time of the day2 (twice a day)basalnph, idet, iglarat bedtime and in the morningpremixedbhi, biasp, lispromixwith major meals coformulationideg aspwith major meals basal plusbasal + prandialat bedtime + with major meal3 (thrice a day)prandialregular, aspart, lispro, glulisinewith mealsbolus coformulationaspart + idegaspwith meals4 or 5 (four or five times a day)basal bolusany combination of basal and boluswith meals, and at bedtime or twice dailycsi (continuous insulin infusion pump)alternative to multiple injection antipodal meal (i.e., meals spaced roughly 12 h apart) minimum 8-h gap between 2 dosestable 2prescription patterns of currently available insulin preparations, supported by evidence from various published clinical studiesinsulin preparation / frequency of injectionsonce a daytwice a daythrice a daymore than thrice a dayinsulin degludeczinman et al. t1 dm patients (n = 629) nananainsulin glargine u 300riddle et al. edition i. t2 dm (n = 807) yki - jarvinen et al. am diabetes assoc 2014 t1 dm (n = 408) nanainsulin degludec + insulin aspartonishi et al. intensify premix i. t2 dm (n = 446) nanahirsch et al. t2 dm (n = 172) jia et al. lancet diabetes endocrinol 2015. t1 dm (n = 26) insulin aspartmathieu et al. diabetes obes metab 2014. t2 dm (n = 680) nanana basal nph, glargine, detemir, degludec, bhi biphasic human insulin, biasp biphasic insulin aspart, lispromix biphasic insulin lispro, iasp insulin aspart, ideg insulin degludec, idegasp insulin degludec and insulin aspart, ideglira insulin degludec and liraglutide, lixilan lixisenatide and insulin glargine, idet insulin detemir, iglar insulin glargine, iglu insulin glulisine, nph neutral protamine hagedorn, prandial regular, lispro, aspart, glulisine, na not applicable insulin preparations that are currently on the market, along with the prescription patterns for them antipodal meal (i.e., meals spaced roughly 12 h apart) minimum 8-h gap between 2 doses prescription patterns of currently available insulin preparations, supported by evidence from various published clinical studies basal nph, glargine, detemir, degludec, bhi biphasic human insulin, biasp biphasic insulin aspart, lispromix biphasic insulin lispro, iasp insulin aspart, ideg insulin degludec, idegasp insulin degludec and insulin aspart, ideglira insulin degludec and liraglutide, lixilan lixisenatide and insulin glargine, idet insulin detemir, iglar insulin glargine, iglu insulin glulisine, nph neutral protamine hagedorn, prandial regular, lispro, aspart, glulisine, na not applicable newer ultralong - acting basal insulins and coformulations of ultralong - acting insulin analogues with either rapid - acting insulin analogues, or with glp-1ra (glucagon - like peptide-1 receptor agonists), have recently been introduced. while these newer preparations are a combination of two preparations, they definitely do not fit into the earlier category of premixed insulins. other molecules, such as pegylated lispro, are also in advanced stages of development, and will soon be available for clinical use. basal insulins were initially thought to be used once a day. as nph, glargine, and detemir do not provide adequate 24 h coverage, they may need to be used twice daily in certain patients, especially those with type 1 diabetes. the novel ultralong - acting insulin degludec provides adequate 24-h glycemic control and can be used once daily at any time of the day. these factors need to be reflected in an updated taxonomic profile of insulin. while basal insulins are able to achieve adequate fasting control in many cases, they are unable to provide prandial coverage. initiation of a once - daily premix or coformulation with the major meal or meal with highest glycemic excursion allows control of postprandial glucose after one meal as well. the frequency of administration of these insulin preparations can, if required, be intensified to twice or thrice daily. while biphasic human insulin or premixed analogue insulin need to be administered at antipodal meals (i.e., meals spaced roughly 12 h apart), idegasp (insulin degludec aspart) may be administered at two consecutive meals, provided an 8-h gap is maintained. all of these patterns of use find a place in a number - based umbrella of insulin taxonomy, as opposed to the traditional regime classification, which proposes only twice daily use of premixed insulin. if the twice - daily regime does not achieve 24-h euglycemia, intensive insulin therapy (defined as that including 3 or more than 3 injections per day) may be required in the form of either three premix insulin injections or a basal bolus regimen. depending upon the needs of the patient, one can prescribe prandial insulin thrice a day; premixed twice and prandial once; or prandial twice and premixed / coformulation once. basal bolus regimes involving 3 bolus doses and 1 or 2 basal doses can also be used in refractory patients and in type 1 diabetes. the number - based taxonomy is able to include all of these regimes as subclasses (table 1), based upon published randomized controlled trials (table 2). this arrangement makes it much simpler for the student to understand the subject of insulin pharmacotherapeutics. it helps the practitioner to appreciate the versatility of insulin and the many ways in which this life - saving molecule can be used. this system also allows the physician to choose the appropriate regime for a particular patient while following person - centeredness in letter and spirit. at the same time, choice of regime should take biomedical factors such as severity of hyperglycemia, risk of hypoglycemia, and diabesity indices into account. such a codification would promote appropriate choice of therapy based upon the individual s glucophenotype, motivation level, and psychosocial limitations, ease of use, and acceptance of insulin, by sensitizing the diabetes care professional to the patient s needs. we therefore propose that future guidelines and recommendations utilize this person - centered arrangement of insulin regimes, rather than straitjacketing preparations according to traditional criteria.

this article describes a number - based system for the classification of insulin regimes. it utilizes a patient - centered variable (number of injections per day) and pharmacokinetic / dynamic characteristics to craft a taxonomic system that is able to incorporate all available insulin preparations and coformulations. this framework of systematics is robust enough to include various molecules that have been recently developed. it serves to enhance understanding of the subject, and facilitates the practical or clinical usage of theoretical knowledge. we propose that number - based insulin taxonomic models should be used in clinical guidelines and recommendations rather than restricting ourselves to pharmaceutical - based classifications. pubmed articles including both review articles and clinical trials published since the year 1990 were searched, to gather evidence and information on the various types of insulins available, and how they can be used, based on the number or frequency of injections prescribed per day.

honey has been reported to show a significant antibacterial activity against a wide range of bacteria. it has been demonstrated in many studies that the antibacterial effects of honey are attributed to its high osmolarity, low ph, hydrogen peroxide content, and other non - peroxide factors such as lysozyme, phenolic acids, and flavonoids. the quality of honey is mainly determined by its sensorial, chemical, physical, and microbiological characteristics. honey is a sweet and flavorful natural product which has been consumed for its high nutritive value and its contribution to human health that depend largely on the floral source. these properties could be associated to honey's high osmolarity, antibacterial properties, and antioxidant capacity. the antioxidant and antibacterial activity of honey, however, varies greatly depending on the honey floral source. there are about 50 species in the tropical asia, africa, america, and the temperate regions. in flora iranica , five species were listed for iranian plateau, namely zizyphus spina - christi, zizyphus oxyphylla nummularia, zizyphus oxyphylla jujuba, zizyphus oxyphylla mauritiana, and zizyphus oxyphylla. z. spina - christi l. known by the persian name biological and pharmacological tests have shown antibacterial, antiviral, and antidiabetic effects of the extracts or fractions of the leaves of this plant. the honey collected from the flowers of the sidr is in high demand by citizens for its medicinal qualities in addition to its excellent taste and fragrant smell. the composition of active components in plants depends on various factors, particularly plant bio- and chemo type and climatic conditions. consequently, it can be reasonably expected that honey properties from different locations are different. newly identified honeys may have advantages over or similarities with known honey due to enhanced antimicrobial activity, local production (thus availability), and/or greater selectivity against medically important organisms. on this basis, therefore, the aim of the present study was an in vitro evaluation of antibacterial effectiveness of ziziphus honey originating from the z. spina - christi l. tree (tangestan, bushehr) against listeria monocytogenes, salmonella typhimurium, escherichia coli, and staphylococcus aureus as foodborne pathogens. s. aureus (atcc 6538) and l. monocytogenes (atcc 19118) as gram - positive and s. typhimurium (atcc 14028) and e. coli (atcc 25922) as gram - negative bacteria were used. for the antimicrobial activity measurement, first of all active cultures were generated by inoculating single colony of each bacterium into 5 ml sterile nutrient broth (merck) and incubated at 37c for 24 h. freshly synchronized cultures of bacterial strains from initial inoculums were prepared after 2 times overnight cultures (24 h) of each bacterium by successively transferring 100 l of the vegetative cells into tryptic soy broth (tsb, merck, nj, usa). then the optical densities of the active freshly synchronized cultures were adjusted at 600 nm. the broth cultures prepared in this way had a final culture density of approximately 10 colony forming unit (cfu)/ml. this had been used as adjusted inoculum for all the further studies. natural ziziphus honey was collected from tangestan apiaries in bushehr province, iran and stored in the dark at room temperature. different concentrations of honey constituting 10%, 20%, 30 %, and 40% (v / v) were made using sterile nutrient broth. before, honey was placed in a 37c water bath to aid dissolving. the natural ziziphus honey in 10%, 20%, 30%, and 40% dilutions (v / v) were provided in nutrient broth medium as mentioned above. then, 1 ml (about 10 cfu / ml) of each of bacteria separately added to 1 ml of each dilution of honey and incubated at 35c for 120 h. concurrently, a positive control by inoculating 1 ml of the microbial suspension into 1 ml of the nutrient broth and a negative control by adding 1 ml of the nutrient broth (without bacteria) to 1 ml of each dilution of honey were made. total viable count enumeration of each sample was investigated after 0, 24, 72, and 120 h postinoculation. total viable count was carried out using the pour - plate method as described by harrigan. all tests were performed in triplicate and were repeated 3 times to obtain reliable . statistical analysis of data was performed using analysis of variance (anova) and post - hoc tests (scheffe and duncan) by spss 16 software from ibm company. s. aureus (atcc 6538) and l. monocytogenes (atcc 19118) as gram - positive and s. typhimurium (atcc 14028) and e. coli (atcc 25922) as gram - negative bacteria were used. for the antimicrobial activity measurement, first of all active cultures were generated by inoculating single colony of each bacterium into 5 ml sterile nutrient broth (merck) and incubated at 37c for 24 h. freshly synchronized cultures of bacterial strains from initial inoculums were prepared after 2 times overnight cultures (24 h) of each bacterium by successively transferring 100 l of the vegetative cells into tryptic soy broth (tsb, merck, nj, usa). then the optical densities of the active freshly synchronized cultures were adjusted at 600 nm. the broth cultures prepared in this way had a final culture density of approximately 10 colony forming unit (cfu)/ml. this had been used as adjusted inoculum for all the further studies. natural ziziphus honey was collected from tangestan apiaries in bushehr province, iran and stored in the dark at room temperature. different concentrations of honey constituting 10%, 20%, 30 %, and 40% (v / v) were made using sterile nutrient broth. before, the natural ziziphus honey in 10%, 20%, 30%, and 40% dilutions (v / v) were provided in nutrient broth medium as mentioned above. then, 1 ml (about 10 cfu / ml) of each of bacteria separately added to 1 ml of each dilution of honey and incubated at 35c for 120 h. concurrently, a positive control by inoculating 1 ml of the microbial suspension into 1 ml of the nutrient broth and a negative control by adding 1 ml of the nutrient broth (without bacteria) to 1 ml of each dilution of honey were made. total viable count enumeration of each sample was investigated after 0, 24, 72, and 120 h postinoculation. total viable count was carried out using the pour - plate method as described by harrigan. all tests were performed in triplicate and were repeated 3 times to obtain reliable . statistical analysis of data was performed using analysis of variance (anova) and post - hoc tests (scheffe and duncan) by spss 16 software from ibm company. viable count enumeration of different concentrations of ziziphus honey (10%, 20%, 30%, and 40%) was investigated after 0, 24, 72, and 120 h postinoculation with any of the bacteria. based on our and statistical analysis using one - way anova, significant difference at different times between different concentrations of honey was observed about all the researched bacteria. the were expressed as means standard deviation log - transformed bacterial concentration (cfu / ml). figure 1 indicates antibacterial activity of ziziphus honey against s. aureus at different concentrations and times. the showed 3 - 7 log reduction of s. aureus in 30% and 40% concentration of honey, respectively, at different times. also in post - hoc tests (scheffe and duncan) in 24 h of incubation was observed significant difference between the concentrations of 0% with the other concentrations of honey (p < 0.05). on the other hand, between the concentrations of 10%, 20%, and 30% showed no any significant difference (p > 0.05). in 120 h of incubation, the concentrations of 30% and 40% were observed significant difference compared to other concentrations (p < 0.05). the statistical analysis showed no significant difference between 24, 72, and 120 h incubation on s. aureus in 40% dilution. antibacterial activity of ziziphus honey against e. coli indicates 3 - 7 log reduction in different concentrations of honey, after 120 h of incubation. statistical analysis observed a significant difference between concentrations of 30% and 40% with other concentrations at 24 and 72 h of incubation (p < 0.05). based on the , l. monocytogenes showed 2 - 5 log reduction in different concentrations of honey, after 72 and 120 h of incubation. also, a significant difference between different concentrations of honey with 0% concentration was observed at 72 and 120 h of incubation (p < 0.05). the effect of ziziphus honey against s. typhimurium showed 1.5 - 6 log reduction in different concentrations of honey, after 72 and 120 h of incubation; also, a significant difference between different concentrations of honey with 0% concentration was observed at 72 and 120 h of incubation (p < 0.05). statistical analysis between different concentrations indicated a significant difference between 30% and 40% concentrations of honey with other concentrations after 24 h of incubation (p < 0.05). also, it showed no significant difference between 30% and 40% dilution on salmonella (p > 0.05). the total showed that ziziphus honey has a good antibacterial activity against these bacteria. in a comparative trial, antibacterial activity of ziziphus honey was higher after 120 h incubation, especially in 30% and 40% concentrations of honey. growth curve of staphylococcus aureus in different dilutions (10%, 20%, 30%, and 40%) in 0, 24, 72, and 120 h growth curve of escherichia coli in different dilutions (10%, 20%, 30%, and 40%) in 0, 24, 72, and 120 h growth curve of listeria monocytogenes in different dilutions (10%, 20%, 30%, and 40%) in 0, 24, 72, and 120 h growth curve of salmonella typhimurium in different dilutions (10%, 20%, 30%, and 40%) in 0, 24, 72, and 120 h these days, abundant use of antibiotics has ed in widespread resistance; with the development of novel antibiotics, alternative antimicrobial strategies are urgently needed. honey has an extensive history to traditional human medicine use and also is an attractive ingredient for healthy foods. it has been proposed that the healing effect of honey could be due to various physical and chemical properties. the high osmolarity and acidity of honey are among the physical characteristics that contribute to its antibacterial activity. hydrogen peroxide, volatiles, organic acids, flavonoids, beeswax, nectar, pollen, and propolis are important chemical factors that provide antibacterial properties to honey. in a study, shin and ustunol related the sugar composition of honeys from different floral sources to the growth inhibition of various intestinal bacteria. thus, the floral source of honey plays an important role on its biological properties. the composition of honey has been shown to depend largely on its floral source. in consequence , it would not be surprising that the provenance of honey could determine its antibacterial properties. z. spina - chirsti plant has versed medicinal and nutritional values. some useful phytochemicals that include flavonoids, tannins, lipids, terpenes, alkaloids, steroids, free sugar, and mucilage have been isolated from the plant. this research is the first study to provide direct evidence for the efficacy of iranian ziziphus honey against these foodborne pathogens. our data reveal that the foodborne pathogens tested were susceptible to ziziphus honey, although this honey was not able to produce complete inhibition of bacterial growth during 120 h incubation, but antibacterial activity was increased with additional concentration of honey and the microbial count showed about 3 - 7.5 log reduction after 120 h in most cases. nzeako and hamdi in their study of six commercial honeys found that inhibition of s. aureus, e. coli, and pseudomonas aeruginosa did not occur at honey concentrations < 40%. in a research in saudi arabia, antimicrobial effects of different honey samples including sidr honey against multiresistant pathogens showed that the most sensitive pathogens were aspergillus nidulans, s. typhimurium, and staphylococcus epidermidis. alandejani et al. showed effectiveness of sidr (yemen) and manuka (new zealand) honey on s. aureus and p. aeruginosa biofilms, respectively. the bactericidal rates for the sidr and manuka honeys against methicillin - susceptible s. aureus, methicillin - resistant s. aureus, and p. aeruginosa biofilms were 63 - 82%, 73 - 63%, and 91 - 91%, respectively. these rates were significantly higher than those seen with single antibiotics commonly used. in a research, hegazi investigated antibacterial activity of seven egyptian honeys and one saudi honey (sidr) against some bacteria. he reported that the sidr honey was more effective against s. aureus and p. aeruginosa. also, antimicrobial activity of three types of honey (sidr, sunflower, and sunut) was examined in sudan. the sidr honey showed antimicrobial activity against s. aureus, p. aeruginosa, and klebsiella aerogenes, while e. coli showed high resistance. other researchers have also found differences in susceptibility of microorganisms to ziziphus (sidr) honey produced in different countries. there is a great interest in controlling the growth or eliminating foodborne pathogens using natural antimicrobials. if honey can slow or stop the growth of spoilage organisms or food pathogens, then its incorporation into foods as a preservative can be explored. in light of the enormous potential for application of honey, it is important that research continues not only into those honeys recognized as antibacterial, but also into other locally produced, as yet untested honeys. therefore, it is recommended using ziziphus honey as a natural preservative and antibacterial agent and it may play an important role as antibacterial natural product. however, further research could indicate whether ziziphus honey has potential as a preservative in foods and oral medications.

: honey has previously been shown to have wound healing and antimicrobial properties, but this is dependent on the type of honey, geographical location, and flower from which the final product is derived. we tested the antimicrobial activity of a natural honey originating from the ziziphus spina - christi tree, against selected strains of bacteria. ziziphus honey among more than a 100 verities of honey is known to have the greatest value of energy and minerals in it.materials and methods: the aim of this study was to determine the antibacterial activity of ziziphus honey in 10%, 20%, 30%, and 40% dilutions (v / v) against listeria monocytogenes, salmonella typhimurium, escherichia coli, and staphylococcus aureus. viable count enumeration of the sample was investigated after 0, 24, 72, and 120 h postinoculation with any of the bacteria using pour - plate method.:the findings indicate that ziziphus honey was effective against these pathogenic bacteria. in a comparative trial, antibacterial activity of ziziphus honey was higher after 120 h incubation for each four bacteria in most dilutions. the microbial count showed 3 - 7.5 log reduction comparing with control after 120 h.:therefore, it is recommended using ziziphus honey as a natural preservative and antibacterial agent. also, it could potentially be used as therapeutic agents against bacterial infection particularly to the tested microorganisms.

diphthamide, the target of diphtheria toxin, is a unique posttranslational modification on translation elongation factor 2 (ef2) in archaea and eukaryotes. the biosynthesis of diphthamide was proposed to involve three steps. the first step is the transfer of the 3-amino-3-carboxypropyl group from s - adenosyl - l - methionine (sam) to the histidine residue of ef2, forming a c c bond. previous genetic studies showed this step requires four proteins in eukaryotes, dph1dph4. however, the exact molecular functions for the four proteins are unknown. previous study showed that pyrococcus horikoshii dph2 (phdph2), a novel iron - sulfur cluster - containing enzyme, forms a homodimer and is sufficient for the first step of diphthamide biosynthesis in vitro. here we demonstrate by in vitro reconstitution that yeast dph1 and dph2 form a complex (dph1-dph2) that is equivalent to the homodimer of phdph2 and is sufficient to catalyze the first step in vitro in the presence of dithionite as the reductant. we further demonstrate that yeast dph3 (also known as kti11), a csl - type zinc finger protein, can bind iron and in the reduced state can serve as an electron donor to reduce the fe - s cluster in dph1-dph2. our study thus firmly establishes the functions for three of the proteins involved in eukaryotic diphthamide biosynthesis. for most radical sam enzymes in bacteria, flavodoxins and flavodoxin reductases are believed to serve as electron donors for the fe - s clusters. the finding that dph3 is an electron donor for the fe - s clusters in dph1-dph2 is thus interesting and opens up new avenues of research on electron transfer to fe - s proteins in eukaryotic cells.

the prostamide bimatoprost 0.03% and the prostaglandin analogs travoprost 0.004% and latanoprost 0.005% are ocular hypotensive lipids, the newest class of drugs indicated for the lowering of intraocular pressure (iop) in patients with glaucoma and ocular hypertension. these medications, administered once daily, have greater iop - lowering efficacy than the beta - blocker timolol.13 due to the importance of even small increments of iop reduction for preservation of visual function among those with glaucoma or ocular hypertension, patients who are not achieving sufficiently low iop with their current medications may benefit from trying a new regimen. to avoid delay in determining the effect of such changes, a paired - eye comparison was used to assess the efficacy of prostaglandin / prostamide medications in patients who had uncontrolled or poorly controlled iop. this retrospective chart review of patients participating in individual, within - practice, paired - eye, open - label comparisons included consecutive patients seen at a private practice clinic between september 2002 and may 2005. patients had a diagnosis of glaucoma or ocular hypertension and their iop had been uncontrolled in the investigator s clinical judgment. patients had been clinically judged to be suitable candidates for therapy with agents of the prostamide / prostaglandin class. the three medications studied in this evaluation were bimatoprost 0.03% (lumigan ; allergan, inc ., irvine, ca), travoprost 0.004% (travatan ; alcon laboratories, inc ., fort worth, tx), and latanoprost 0.005% (xalatan, pfizer, inc . patients were assigned to bimatoprost in one randomly assigned eye and another medication in the fellow eye . the second medication was determined at random, except for patients who were already using one of the prostaglandin / prostamide analogs . patients included in this evaluation were examined by the physician and had iop measured at the initial visit . per clinical routine, patients were scheduled for a follow - up visit 46 weeks later, when iop was measured and hyperemia or other side effects were recorded . at the follow - up visit, patients were asked about the eye drops in a general way such as, how are you doing with the new drops ? or how are your new drops ? the physician then discussed with each patient the iop findings for each eye and other aspects of the treatment . after conferring with the physician, patients determined in some cases, a reason for the patient s choice was noted in the chart . patient charts were excluded from analysis if they had any ocular surgery between the initial and follow - up visit . the main outcome measures of the chart review were mean change in iop from baseline, and percentage of patients choosing a particular medication for continued use . unpaired t - tests were used to evaluate the statistical significance of iop changes from baseline within a treatment group, and paired t - tests were performed to determine statistical significance among treatment groups in the bilateral comparison . a total of 91 patients participated in the paired - eye comparison . of these, 36 were excluded from the chart review analysis ; 17 patients charts were not available at the time of data collection, seven did not have follow - up data, four did not have a confirmatory diagnosis, three did not receive bimatoprost in one of the eyes, two had eye surgery between the baseline and follow - up visit, two patients did not make a clear choice and proceeded to use both study agents, and one individual only used one of the comparative agents . fifty patients received bimatoprost in one eye and travoprost in the other eye, and five patients received bimatoprost in one eye and latanoprost in the other eye . bimatoprost was randomized to the right eye in 30 patients and to the left eye in 25 patients ; travoprost was randomized to the right eye in 22 patients and to the left eye in 28 patients ; and latanoprost was randomized to the right eye in three patients and to the left eye in two patients . seventy - five percent of patients were taking at least one glaucoma medication, with 42% of patients on monotherapy and 35% ( 19/55) using two or more medications (table 2). because the majority of patients (58%, 32/55) were using latanoprost prior to enrolling in the study (table 2), few participants were assigned to receive latanoprost during the study. the median interval between the baseline and follow - up visits was 42 days (mean 72.7 days), and 10 patients had their follow - up visit more than 90 days after the baseline visit. data were obtained for all outcome measures for all patients, except for three patients receiving bilateral bimatoprost or travoprost, for whom iop - lowering data were not documented in the medical chart at the follow - up visit. bimatoprost and travoprost significantly reduced iop from baseline during the study period (figure 1). for eyes receiving bimatoprost with documented iop measurement at baseline and at follow - up (n = 52), iop decreased by 2.7 mmhg (standard deviation = 4.2 mmhg), a significant reduction (p < 0.0001) versus baseline. mean (standard deviation) baseline iop for bimatoprost - treated eyes was 19.8 4.9 mmhg (range 1034) and dropped to 17.1 4.9 mmhg (range 1937) at the end of the study period. for travoprost - treated eyes with documented iop measurement at baseline and at follow - up (n = 47), iop was reduced from a baseline value of 19.4 4.1 mmhg (range 1231) to 17.7 4.2 mmhg (range 929) at the end of the study period, for a decrease of 1.7 mmhg (p = 0.009 versus baseline). the numeric magnitude of mean iop reduction with bimatoprost was larger than with travoprost (2.7 mmhg versus 1.7 mmhg decrease), but the difference did not reach statistical significance (p = 0.230). in eyes receiving latanoprost (n = 5), mean baseline iop was 19.6 4.7 mmhg (range 1526), and iop at follow - up averaged 16.2 2.5 mmhg (range 1420), for a reduction of 3.4 3.2 mmhg. this difference was not statistically significant (p = 0.077), probably because of the small sample size. at the follow - up visit, the physician discussed the clinical outcomes with each patient, and each patient chose which medication they would prefer to continue using. patients who received bimatoprost in one eye and travoprost in the fellow eye chose to continue with bimatoprost over travoprost by a factor of 2.4 to 1 (figure 2). nearly seven of 10 patients chose bimatoprost (68%, 34/50), 28% of patients (14/50) chose travoprost, and 4% (2/50) chose neither. the two patients who chose neither option had used latanoprost prior to the study, and elected to continue with it afterwards. among the five patients who received bilateral bimatoprost or latanoprost, three chose to continue with bimatoprost, one chose latanoprost, and one chose neither (this patient had been using travoprost prior to the study and chose to continue with it). table 3 shows the primary factor that patients stated influenced their choice of medication. among the 15 patients who stated a reason, most (80%, 12/15) cited improvement in iop as the primary factor in their decision. a majority of these patients (83%, 10/12) preferred bimatoprost over the agent they had used in the fellow eye. other reasons offered by patients for determination of medication choice were pressure change plus better side effect profile , convenience , and price , cited by one patient each (medications chosen were travoprost, and latanoprost, respectively). no patients cited side effect profile as a primary influence on their medication choice. looking at the entire study population analyzed (n = 55), hyperemia was reported by eight patients, with six reporting hyperemia in both eyes, one reporting hyperemia in the bimatoprost - treated eye only, and one reporting hyperemia in the travoprost - treated eye only. a total of 91 patients participated in the paired - eye comparison. of these, 36 were excluded from the chart review analysis; 17 patients charts were not available at the time of data collection, seven did not have follow - up data, four did not have a confirmatory diagnosis, three did not receive bimatoprost in one of the eyes, two had eye surgery between the baseline and follow - up visit, two patients did not make a clear choice and proceeded to use both study agents, and one individual only used one of the comparative agents. fifty patients received bimatoprost in one eye and travoprost in the other eye, and five patients received bimatoprost in one eye and latanoprost in the other eye. bimatoprost was randomized to the right eye in 30 patients and to the left eye in 25 patients; travoprost was randomized to the right eye in 22 patients and to the left eye in 28 patients; and latanoprost was randomized to the right eye in three patients and to the left eye in two patients. seventy - five percent of patients were taking at least one glaucoma medication, with 42% of patients on monotherapy and 35% (19/55) using two or more medications (table 2). because the majority of patients (58%, 32/55) were using latanoprost prior to enrolling in the study (table 2), few participants were assigned to receive latanoprost during the study. the median interval between the baseline and follow - up visits was 42 days (mean 72.7 days), and 10 patients had their follow - up visit more than 90 days after the baseline visit. data were obtained for all outcome measures for all patients, except for three patients receiving bilateral bimatoprost or travoprost, for whom iop - lowering data were not documented in the medical chart at the follow - up visit. bimatoprost and travoprost significantly reduced iop from baseline during the study period (figure 1). for eyes receiving bimatoprost with documented iop measurement at baseline and at follow - up (n = 52), iop decreased by 2.7 mmhg (standard deviation = 4.2 mmhg), a significant reduction (p < 0.0001) versus baseline. mean (standard deviation) baseline iop for bimatoprost - treated eyes was 19.8 4.9 mmhg (range 1034) and dropped to 17.1 4.9 mmhg (range 1937) at the end of the study period. for travoprost - treated eyes with documented iop measurement at baseline and at follow - up (n = 47), iop was reduced from a baseline value of 19.4 4.1 mmhg (range 1231) to 17.7 4.2 mmhg (range 929) at the end of the study period, for a decrease of 1.7 mmhg (p = 0.009 versus baseline). the numeric magnitude of mean iop reduction with bimatoprost was larger than with travoprost (2.7 mmhg versus 1.7 mmhg decrease), but the difference did not reach statistical significance (p = 0.230). in eyes receiving latanoprost (n = 5), mean baseline iop was 19.6 4.7 mmhg (range 1526), and iop at follow - up averaged 16.2 2.5 mmhg (range 1420), for a reduction of 3.4 3.2 mmhg. this difference was not statistically significant (p = 0.077), probably because of the small sample size. at the follow - up visit, the physician discussed the clinical outcomes with each patient, and each patient chose which medication they would prefer to continue using. patients who received bimatoprost in one eye and travoprost in the fellow eye chose to continue with bimatoprost over travoprost by a factor of 2.4 to 1 (figure 2). nearly seven of 10 patients chose bimatoprost (68%, 34/50), 28% of patients (14/50) chose travoprost, and 4% (2/50) chose neither. the two patients who chose neither option had used latanoprost prior to the study, and elected to continue with it afterwards. among the five patients who received bilateral bimatoprost or latanoprost, three chose to continue with bimatoprost, one chose latanoprost, and one chose neither (this patient had been using travoprost prior to the study and chose to continue with it). table 3 shows the primary factor that patients stated influenced their choice of medication. among the 15 patients who stated a reason, most (80%, 12/15) cited improvement in iop as the primary factor in their decision. a majority of these patients (83%, 10/12) preferred bimatoprost over the agent they had used in the fellow eye. other reasons offered by patients for determination of medication choice were pressure change plus better side effect profile , convenience , and price , cited by one patient each (medications chosen were travoprost, and latanoprost, respectively). no patients cited side effect profile as a primary influence on their medication choice. looking at the entire study population analyzed (n = 55), hyperemia was reported by eight patients, with six reporting hyperemia in both eyes, one reporting hyperemia in the bimatoprost - treated eye only, and one reporting hyperemia in the travoprost - treated eye only. patients with glaucoma or ocular hypertension who did not have sufficient iop control with their current medication regimens instilled bimatoprost in one eye and either travoprost or latanoprost in the other eye. significant iop reductions were observed for bimatoprost- and travoprost - treated eyes, consistent with published .14 relatively few studies have directly compared the efficacy of prostaglandin / prostamide glaucoma therapies. in a meta - analysis of all head - to - head randomized, investigator - masked direct comparisons of latanoprost, bimatoprost, and travoprost, bimatoprost was more efficacious at lowering iop than travoprost in the two direct comparisons identified (involving 150 patients), and bimatoprost was found to be more efficacious than latanoprost in six head - to - head studies identified (involving 460 patients).4 our study may not have been adequately powered to detect statistically significant differences among therapies. however, for the two therapies where we detected a significant iop decline from baseline (bimatoprost and travoprost), bimatoprost showed qualitatively greater iop reduction than travoprost, consistent with a report by holmstrom et al.4 an important limitation of this assessment was the fact that relatively few eyes were randomized to receive latanoprost. because nearly 60% of study participants had been receiving latanoprost prior to the study, only five patients received latanoprost as one of the comparators. therefore, for these patients, the bilateral comparison was limited to travoprost and bimatoprost. because of the small sample size, the with latanoprost may be difficult to generalize. use of a larger study population, or employment of a multiple - study site design in future trials may in a patient population that is more evenly distributed among the cohorts. it should be noted that our are consistent with those of a large - scale real - world study, in which more than 15,000 glaucoma patients on latanoprost were switched to bimatoprost in a nationwide health maintenance organization.5 in that study, patients switching to bimatoprost experienced statistically significant iop reduction, and few reverted to their previous medication.5 nonadherence to glaucoma treatment regimens is a significant problem, in large part because glaucoma is chronic, progressive, and symptom - free in its early stages.68 in the treatment practice described in this report, patients chose which medication to continue using after consulting with the physician. physician partnership in medical decision - making is increasing as patients become more empowered through consumerism and access to independent information resources, such as the internet.9 a majority of patients in this study who gave a reason for their choice cited iop - lowering efficacy, as opposed to side effects or other aspects of the medication immediately perceptible to the patient. our , while preliminary, raise the intriguing question of whether patients who are involved in their medical decision - making might also have improved adherence to their glaucoma regimens. after direct comparisons between glaucoma drugs trialed in each eye, patients preferred bimatoprost over travoprost by a factor of 2.4 to 1. today s physicians may wish to consider a simple bilateral trial such as ours for their glaucoma patients who are not achieving adequate iop control with their current medication regimens.

objectiveto evaluate efficacy and patient preference retrospectively among intraocular pressure (iop)-lowering prostamide and prostaglandin medications in a real - world clinical setting.methodschart review of patients with uncontrolled glaucoma or ocular hypertension seen at a private practice clinic (n = 55) who received bimatoprost 0.03% once daily in one eye and either travoprost 0.004% or latanoprost 0.005% once daily in the fellow eye. iop was evaluated at the initial visit and at a follow - up visit scheduled 46 weeks later. at the follow - up visit, each patient discussed the clinical with their physician, chose which medication they preferred to continue using, and were queried regarding the reason for their choice. this paired - eye comparison method is used routinely in clinical practice to assess clinical response and involve patients in decisions regarding treatment. change in mean iop from baseline and patient medication choice were the outcome measures.bimatoprost-treated eyes (n = 52) had a mean iop reduction of 2.7 mmhg and travoprost - treated eyes (n = 47) had an average decrease of 1.7 mmhg (p = 0.230). bimatoprost significantly reduced mean iop (from 19.8 mmhg at baseline to 17.1 mmhg at follow - up, p < 0.0001), as did travoprost (from 19.4 mmhg at baseline to 17.7 mmhg at follow - up, p = 0.009). latanoprost - treated eyes were excluded from the efficacy analysis due to small sample size (n = 5). for continued therapy, patients chose bimatoprost over travoprost by a factor of 2.4 to 1. of the 15 patients who gave a reason for their choice, 80% said their decision was based primarily on iop change.bimatoprost and travoprost were efficacious in reducing iop among patients with uncontrolled glaucoma and ocular hypertension. patients preferred bimatoprost over travoprost when trialed in fellow eyes.

personalized medicine has gained a lot of attention in recent years with improved understanding of the contribution of genetic factors to benign and malignant diseases and responses to pharmacological interventions, and the availability of a wide spectrum of genetic tests at low costs. although the term " personalized medicine " is often used to refer to individualizing care according to the genetic make - up of the patient, this term can be interpreted more broadly to mean " tailoring care to the individual patient characteristics ". in other words, personalized medicine can be interpreted as an approach to classify individuals into subpopulations that differ in their susceptibility to a particular disease, rate of disease progression, and response to specific treatments such that preventative or therapeutic interventions can be concentrated on those who will benefit, sparing expense and side effects for those who will not. simply put, the goal of personalized medicine is to provide optimal care according to the individual patient's disease characteristics, personal preference, comorbidities, and social circumstances such that maximum benefit can be derived while minimizing costs and adverse reactions. substantial progress has been made in the treatment of hepatitis b in the last 15 years. there are currently seven approved drugs for the treatment of hepatitis b: two formulations of interferon (ifn) - conventional and pegylated ifn (peg - ifn), and five nucleos(t)ide analogues - lamivudine, telbivudine, adefovir, entecavir, and tenofovir. these drugs can suppress hepatitis b virus (hbv) replication, decrease hepatic inflammation and fibrosis and even reverse cirrhosis, prevent complications of cirrhosis, and reduce the incidence of hepatocellular carcinoma (hcc).1,2,3 however, currently approved drugs do not eradicate hbv and have low rates of clearance of hepatitis b e antigen (hbeag) and hepatitis b surface antigen (hbsag) and hcc continues to occur albeit at a lower rate. because currently available treatment has little or no effect on covalently closed circular dna, the template for transcription of hbv pregenomic rna and messenger rna and translation of viral proteins or on restoration of host immune response to hbv, viral relapse occurs in most patients when treatment is stopped. thus, most patients require many years and often lifelong treatment to derive continued benefit. long durations of treatment are associated with increasing risks of adverse reactions, antiviral drug resistance, costs, and nonadherence to medications. thus, a major dilemma in hepatitis b treatment is when to initiate therapy. while all patients with chronic hbv infection are at risk of cirrhosis, liver failure and hcc, not all patients will experience these outcomes. immune control of hbv with spontaneous loss of hbeag and hbsag can occur and patients can remain in remission for many years. professional society guidelines provide frameworks for managing patients with hepatitis b but these guidelines have to be interpreted in the context of the individual patient's clinical and social circumstances. personalized management of hepatitis b can be applied based on prediction of the individual patient's risk of cirrhosis and hcc to guide the frequency and intensity of monitoring including hcc surveillance and urgency of treatment. it can also be applied to decisions on when to start treatment, which drug to use, and when to stop based on the individual patient's disease characteristics, preference, comorbidities and other mitigating circumstances. host, viral and environmental factors contribute to an hbsag - positive person's risk of cirrhosis and hcc (table 1). although several studies have identified genetic markers associated with increased risk of hbv - related hcc, these markers have not been validated in broad patient populations of diverse racial / ethnic origins infected with all hbv genotypes. therefore, genetic markers have not yet been incorporated into prediction models for cirrhosis or hcc. during the past decade , many models have been developed to predict the risk of cirrhosis or hcc in persons with chronic hbv infection. some models incorporate many variables including blood test such as hbv genotype, precore and core promoter variants that are not routinely available or clinical information such as alcohol and tobacco use that are unreliable unless the information is collected using standardized questionnaire. table 2 summarizes three prediction models for hcc based on routinely available clinical and laboratory information: reach - b, cu - hcc, and gag - hcc.4,5,6,7,8 these models can be used to predict 5- and 10- year risk of hcc to guide the frequency of monitoring, the need for hcc surveillance, and the urgency of antiviral treatment. however, there are limitations to these models. these models were derived from cohorts of patients with chronic hepatitis b in asia, who were predominantly infected with hbv genotypes b and c and who did not receive antiviral treatment. thus, these models may not be applicable in other parts of the world where the prevalent hbv genotypes are different and where hbv infection is mostly acquired in adult life and not early childhood or in patients receiving antiviral treatment. indeed, two studies of patients receiving antiviral treatment in europe found that performance of these scores, particularly the reach - b score was poor.9,10 by contrast, one study in hong kong showed that all three models accurately predicted which patients with chronic hepatitis b treated with entecavir will develop hcc.11 the american, asian - pacific and european liver association (aasld, apasl and easl) guidelines recommend that treatment decision should be made based on clinical status, serum hbv dna and alanine aminotransferase (alt) levels, hbeag status, and liver histology if available.12,13,14 all guidelines recommend starting treatment as soon as possible in patients with life - threatening liver disease: acute liver failure, decompensated cirrhosis or severe acute exacerbation of chronic hepatitis b regardless of hbv dna and alt levels. these guidelines also recommend antiviral treatment for patients with compensated cirrhosis regardless of alt levels but there are minor differences in threshold hbv dna levels for initiating treatment. all guidelines agree that treatment should be initiated in noncirrhotic patients with serum hbv dna levels greater than 20,000 iu / ml and persistently elevated alt levels and/or histologic evidence of moderate or severe inflammation or fibrosis. however, cutoff values of hbv dna and alt and the need for liver biopsy or non - invasive assessment of liver fibrosis in determining treatment indications vary slightly among the guide - lines. because hbv dna and alt levels fluctuate during the course of chronic hbv infection, all guidelines agree that serial hbv dna and alt levels are more important than values at a single time point in making treatment decisions. furthermore, all guidelines recommend that patients who are not started on treatment should be monitored such that treatment may be initiated at a later time when hbv replication or liver disease becomes more active. the aasld, apasl and easl guidelines are based on a combination of scientific evidence and expert opinion and experience. these guidelines have not incorporated prediction models for cirrhosis or hcc into recommendations when or which patient should start treatment but incorporation of risk scores for hcc may be appropriate for asian patients particularly those in the gray zone. the guidelines emphasize that patient age, family history of hcc, occupational requirements, plans to start a family (for women), and patient preference should also be considered in making treatment decisions. there are many settings when a patient's medical or social circumstances warrant a personalized approach that may be " at odds " with the guidelines but appropriate for that patient. for example, a 48 year old man who is hbeag - positive with serial alt 19 - 28 u / l and hbv dna 3,000,000 - 400,000,000 iu / ml with no evidence of cirrhosis would be considered to be in the immune tolerant phase and " not meet " criteria for treatment but starting this patient on treatment is appropriate because there is ample data showing that patients who remain hbeag - positive after age 40 and those who have serum hbv dna levels higher than 2,000 iu / ml after more than four decades of infection are at increased risk of cirrhosis and hcc.15,16 treatment would also be appropriate in a 32 year old surgeon who has the same disease characteristics if suppression of viremia is a prerequisite for permission to continue his profession as a surgeon and to perform surgical operations (exposure - prone procedures). treatment might also be appropriate in a 45 year old man who is hbeag negative with alt 25 - 35 u / l and hbv dna 1,800 - 9,500 iu / ml if he has several family members with hcc. other examples of personalized approach being " at odds " with guidelines may involve a decision to defer treatment when guidelines would recommend treatment. for example, a 35 year old woman who is hbeag - negative with alt 55 - 65 u / l and hbv dna 28,000 - 75,000 iu / ml with no evidence of cirrhosis may choose to defer treatment if she wishes to start a family and is concerned about the safety of anti - hbv medications on fetal development. another patient with the same disease characteristics may choose to defer treatment if she does not have insurance coverage for medications, or has contraindications to using interferon and is unwilling to commit to many years of nucleos(t)ide analogue therapy. selection of the first - line anti - hbv drug should be based on the safety and efficacy of the drug, risk of drug resistance, cost of the drug, and patient preference. the main advantages of ifn include a finite duration of treatment and a higher rate of hbeag and hbsag loss but ifn has to be administered parenterally and is associated with a wide range of adverse reactions some of which can be serious. entecavir, telbivudine and tenofovir have more potent antiviral activity, and entecavir and tenofovir have higher barriers to antiviral drug resistance. all guidelines recommend initial treatment with peg - ifn, entecavir or tenofovir as monotherapy.12,13,14 because of cost concerns and the lack of access to tenofovir in some asian countries when the most recent version of the apasl guidelines were released, lamivudine, adefovir or telbivudine were also recommended as first - line drugs in treatment - nave patients.13 ifn is not recommended in patients with acute liver failure, decompensated cirrhosis or severe exacerbations of chronic hepatitis b but it may be used with caution in patients with compensated cirrhosis. while guidelines consider peg - ifn, entecavir and tenofovir as equivalent first - line anti - hbv drugs, in clinical practice, entecavir and tenofovir are used much more often than peg - ifn even in patients with no contraindications to the use of ifn. the decision to choose a nucleos(t)ide analogue over peg - ifn may be based on the patient's preference for an oral drug with minimal adverse reactions over an injectable drug with many potential side effects but it may also be based on the physician's beliefs and bias. although cost - effectiveness studies have been performed, these analyses rely on many assumptions and decision on peg - ifn versus nucleos(t)ide analogues is complicated because the efficacy, tolerability, and duration of these two classes of drugs are very different and the costs of these medications vary widely from one country to another. the first step in deciding which drug to use for treating hepatitis b is to choose between peg - ifn versus nucleos(t)ide analogue. patients' preference for route of administration (injection versus oral), duration of treatment (1 year versus many years), and presence of medical / psychiatric comorbidities that contraindicate use of or decrease tolerance to ifn are the most important deciding factors. a personalized decision on anti - hbv drug should also consider the likelihood of response. in this regard, the predictors of response to peg - ifn and nucleos(t)ide analogues are similar: high alt and low hbv dna levels are the most reliable predictors of response to both peg - ifn and nucleos(t)ide analogues. thus, patients who are more likely to respond to peg - ifn are also more likely to respond to nucleos(t)ide analogues. one predictor of response that is unique to peg - ifn is hbv genotype although this applies mainly to hbeag - positive patients. hbeag - positive patients with genotype a hbv have significantly higher rates of hbeag and hbsag loss compared to patients with genotypes b, c or d.17 thus, hbeag - positive patients with genotype a hbv and no contraindications to use of ifn should be encouraged to receive peg - ifn. interleukin-28b (il28b) polymorphism was shown to be a strong predictor of response to ifn - based therapy for hepatitis c but the association between il28b polymorphism and peg - ifn treatment of hepatitis b is inconsistent. other factors may play a role in personalized decisions regarding choice of anti - hbv drugs. for example, young patients may prefer a finite course of peg - ifn versus many years of nucleos(t)ide analogue treatment. this is particularly true for young women contemplating to start a family in the next few years. once a decision is made to treat with nucleos(t)ide analogues, the main considerations are cost and risk of antiviral drug resistance. if cost is not a concern, tenofovir and entecavir are preferred as these drugs have markedly lower rates of drug resistance (0 - 1% after 5 - 6 years of continuous therapy)18,19 compared to lamivudine, adefovir or telbivudine. tenofovir and entecavir have similary potency and barrier to antiviral resistance, and either drug may be used as first - line treatment in nucleoside - nave patients. because of a low risk of nephrotoxicity, entecavir is preferred in older patients, patients with baseline impaired renal function and those with other medical conditions such as hypertension or diabetes that would increase the risk of renal insufficiency. although telbivudine had been reported to improve renal function,20 the high risk of antiviral drug resistance and the potential for other adverse reactions such as myopathy and polyneuropathy make this a poor choice as first - line treatment.21 tenofovir is effective in suppressing not only wild type hbv but also lamivudine, telbivudine or entecavir resistant hbv and to a slightly lesser extent adefovir resistant hbv; therefore, it is a better choice for patients who had been previously treated with other nucleos(t)ide analogues. tenofovir is also a preferred choice for women of reproductive age because of its safety record in pregnancy.22 aasld, apasl and easl guidelines recommend administration of peg - ifn for 48 - 52 weeks in both hbeag - positive and hbeag - negative patients.12,13,14 there is some variation in recommendations when nucleos(t)ide analogues can be stopped. all guidelines recommend that in hbeag - positive patients, nucleos(t)ide analogues can be stopped when the patient has completed 6 - 12 months consolidation therapy after hbeag seroconversion. the easl guidelines recommend continuing treatment until hbsag loss in patients with advanced fibrosis or cirrhosis to avoid flares associated with viral relapse. the aasld and easl guidelines recommend that in hbeag - negative patients, nucleos(t)ide analogues should be continued until hbsag loss but the apasl guidelines stated that treatment may be withdrawn after completing at least two years treatment with undetectable hbv dna documented on three occasions six months apart. all three guidelines recommend lifelong nucleos(t)ide analogue treatment in patients with cirrhosis before treatment unless they had compensated cirrhosis and had cleared hbsag. in addition to pre - treatment factors, several studies have found decrease in hbsag level after 12 or 24 weeks of peg - ifn treatment to be better predictors of response. specifically, a lack of or a small decline in hbsag level after week 12 or 24 of peg - ifn is associated with 85 - 95% negative predictive value of a response.23 these data suggest that a response - guided stop - rule may be used making peg - ifn therapy more attractive because patients who are unlikely to benefit can be spared from further side effects and costs. a recent cost - effective study showed that response - guided peg - ifn therapy is the most cost - effective approach for hbeag - positive but not hbeag - negative patients.24 thus, hbeag - positive patients with no contraindications, particularly those with genotype a hbv, high alt and low hbv dna levels before treatment who are already predicted to have a high likelihood of response to peg - ifn may be further encouraged to have a trial of peg - ifn, treatment can be stopped after 12 weeks if decline in hbsag is inadequate. a caveat to this approach is that it has not been validated prospectively and different stop rules have been proposed by different authors and for different hbv genotypes. for patients receiving nucleos(t)ide analogue therapy, the likelihood of achieving hbeag seroconversion is 40 - 50% and the likelihood of achieving hbsag loss is 0 - 10% after 5 years of continuous treatment.3 thus, more than half of the hbeag - positive patients and 95% of hbeag - negative patients will require more than 5 years of treatment. a few studies found that while viral relapse (redetection of hbv dna in serum) occurs in all hbeag - negative patients who completed 2 - 5 years treatment and who stopped treatment before hbsag loss, clinical relapse with elevated alt and hbv dna levels > 2,000 iu / ml occurred in only 50% of patients.25,26 these data suggest that hbeag - negative patients who have completed > 2 years treatment may consider stopping treatment if they are unwilling to continue or unable to afford continued treatment. however, this personalized approach must be accompanied by a plan for close monitoring for at least 6 months after discontinuation of treatment such that treatment can be promptly resumed if necessary. host, viral and environmental factors contribute to an hbsag - positive person's risk of cirrhosis and hcc (table 1). although several studies have identified genetic markers associated with increased risk of hbv - related hcc, these markers have not been validated in broad patient populations of diverse racial / ethnic origins infected with all hbv genotypes. therefore, genetic markers have not yet been incorporated into prediction models for cirrhosis or hcc. during the past decade , many models have been developed to predict the risk of cirrhosis or hcc in persons with chronic hbv infection. some models incorporate many variables including blood test such as hbv genotype, precore and core promoter variants that are not routinely available or clinical information such as alcohol and tobacco use that are unreliable unless the information is collected using standardized questionnaire. table 2 summarizes three prediction models for hcc based on routinely available clinical and laboratory information: reach - b, cu - hcc, and gag - hcc.4,5,6,7,8 these models can be used to predict 5- and 10- year risk of hcc to guide the frequency of monitoring, the need for hcc surveillance, and the urgency of antiviral treatment. however, there are limitations to these models. these models were derived from cohorts of patients with chronic hepatitis b in asia, who were predominantly infected with hbv genotypes b and c and who did not receive antiviral treatment. thus, these models may not be applicable in other parts of the world where the prevalent hbv genotypes are different and where hbv infection is mostly acquired in adult life and not early childhood or in patients receiving antiviral treatment. indeed, two studies of patients receiving antiviral treatment in europe found that performance of these scores, particularly the reach - b score was poor.9,10 by contrast, one study in hong kong showed that all three models accurately predicted which patients with chronic hepatitis b treated with entecavir will develop hcc.11 the american, asian - pacific and european liver association (aasld, apasl and easl) guidelines recommend that treatment decision should be made based on clinical status, serum hbv dna and alanine aminotransferase (alt) levels, hbeag status, and liver histology if available.12,13,14 all guidelines recommend starting treatment as soon as possible in patients with life - threatening liver disease: acute liver failure, decompensated cirrhosis or severe acute exacerbation of chronic hepatitis b regardless of hbv dna and alt levels. these guidelines also recommend antiviral treatment for patients with compensated cirrhosis regardless of alt levels but there are minor differences in threshold hbv dna levels for initiating treatment. all guidelines agree that treatment should be initiated in noncirrhotic patients with serum hbv dna levels greater than 20,000 iu / ml and persistently elevated alt levels and/or histologic evidence of moderate or severe inflammation or fibrosis. however, cutoff values of hbv dna and alt and the need for liver biopsy or non - invasive assessment of liver fibrosis in determining treatment indications vary slightly among the guide - lines. because hbv dna and alt levels fluctuate during the course of chronic hbv infection, all guidelines agree that serial hbv dna and alt levels are more important than values at a single time point in making treatment decisions. furthermore, all guidelines recommend that patients who are not started on treatment should be monitored such that treatment may be initiated at a later time when hbv replication or liver disease becomes more active. the aasld, apasl and easl guidelines are based on a combination of scientific evidence and expert opinion and experience. these guidelines have not incorporated prediction models for cirrhosis or hcc into recommendations when or which patient should start treatment but incorporation of risk scores for hcc may be appropriate for asian patients particularly those in the gray zone. the guidelines emphasize that patient age, family history of hcc, occupational requirements, plans to start a family (for women), and patient preference should also be considered in making treatment decisions. there are many settings when a patient's medical or social circumstances warrant a personalized approach that may be " at odds " with the guidelines but appropriate for that patient. for example, a 48 year old man who is hbeag - positive with serial alt 19 - 28 u / l and hbv dna 3,000,000 - 400,000,000 iu / ml with no evidence of cirrhosis would be considered to be in the immune tolerant phase and " not meet " criteria for treatment but starting this patient on treatment is appropriate because there is ample data showing that patients who remain hbeag - positive after age 40 and those who have serum hbv dna levels higher than 2,000 iu / ml after more than four decades of infection are at increased risk of cirrhosis and hcc.15,16 treatment would also be appropriate in a 32 year old surgeon who has the same disease characteristics if suppression of viremia is a prerequisite for permission to continue his profession as a surgeon and to perform surgical operations (exposure - prone procedures). treatment might also be appropriate in a 45 year old man who is hbeag negative with alt 25 - 35 u / l and hbv dna 1,800 - 9,500 iu / ml if he has several family members with hcc. other examples of personalized approach being " at odds " with guidelines may involve a decision to defer treatment when guidelines would recommend treatment. for example, a 35 year old woman who is hbeag - negative with alt 55 - 65 u / l and hbv dna 28,000 - 75,000 iu / ml with no evidence of cirrhosis may choose to defer treatment if she wishes to start a family and is concerned about the safety of anti - hbv medications on fetal development. another patient with the same disease characteristics may choose to defer treatment if she does not have insurance coverage for medications, or has contraindications to using interferon and is unwilling to commit to many years of nucleos(t)ide analogue therapy. selection of the first - line anti - hbv drug should be based on the safety and efficacy of the drug, risk of drug resistance, cost of the drug, and patient preference. the main advantages of ifn include a finite duration of treatment and a higher rate of hbeag and hbsag loss but ifn has to be administered parenterally and is associated with a wide range of adverse reactions some of which can be serious. entecavir, telbivudine and tenofovir have more potent antiviral activity, and entecavir and tenofovir have higher barriers to antiviral drug resistance. all guidelines recommend initial treatment with peg - ifn, entecavir or tenofovir as monotherapy.12,13,14 because of cost concerns and the lack of access to tenofovir in some asian countries when the most recent version of the apasl guidelines were released, lamivudine, adefovir or telbivudine were also recommended as first - line drugs in treatment - nave patients.13 ifn is not recommended in patients with acute liver failure, decompensated cirrhosis or severe exacerbations of chronic hepatitis b but it may be used with caution in patients with compensated cirrhosis. while guidelines consider peg - ifn, entecavir and tenofovir as equivalent first - line anti - hbv drugs, in clinical practice, entecavir and tenofovir are used much more often than peg - ifn even in patients with no contraindications to the use of ifn. the decision to choose a nucleos(t)ide analogue over peg - ifn may be based on the patient's preference for an oral drug with minimal adverse reactions over an injectable drug with many potential side effects but it may also be based on the physician's beliefs and bias. although cost - effectiveness studies have been performed, these analyses rely on many assumptions and decision on peg - ifn versus nucleos(t)ide analogues is complicated because the efficacy, tolerability, and duration of these two classes of drugs are very different and the costs of these medications vary widely from one country to another. the first step in deciding which drug to use for treating hepatitis b is to choose between peg - ifn versus nucleos(t)ide analogue. patients' preference for route of administration (injection versus oral), duration of treatment (1 year versus many years), and presence of medical / psychiatric comorbidities that contraindicate use of or decrease tolerance to ifn are the most important deciding factors. a personalized decision on anti - hbv drug should also consider the likelihood of response. in this regard, the predictors of response to peg - ifn and nucleos(t)ide analogues are similar: high alt and low hbv dna levels are the most reliable predictors of response to both peg - ifn and nucleos(t)ide analogues. thus, patients who are more likely to respond to peg - ifn are also more likely to respond to nucleos(t)ide analogues. one predictor of response that is unique to peg - ifn is hbv genotype although this applies mainly to hbeag - positive patients. hbeag - positive patients with genotype a hbv have significantly higher rates of hbeag and hbsag loss compared to patients with genotypes b, c or d.17 thus, hbeag - positive patients with genotype a hbv and no contraindications to use of ifn should be encouraged to receive peg - ifn. interleukin-28b (il28b) polymorphism was shown to be a strong predictor of response to ifn - based therapy for hepatitis c but the association between il28b polymorphism and peg - ifn treatment of hepatitis b is inconsistent. other factors may play a role in personalized decisions regarding choice of anti - hbv drugs. for example, young patients may prefer a finite course of peg - ifn versus many years of nucleos(t)ide analogue treatment. this is particularly true for young women contemplating to start a family in the next few years. once a decision is made to treat with nucleos(t)ide analogues, the main considerations are cost and risk of antiviral drug resistance. if cost is not a concern, tenofovir and entecavir are preferred as these drugs have markedly lower rates of drug resistance (0 - 1% after 5 - 6 years of continuous therapy)18,19 compared to lamivudine, adefovir or telbivudine. tenofovir and entecavir have similary potency and barrier to antiviral resistance, and either drug may be used as first - line treatment in nucleoside - nave patients. because of a low risk of nephrotoxicity, entecavir is preferred in older patients, patients with baseline impaired renal function and those with other medical conditions such as hypertension or diabetes that would increase the risk of renal insufficiency. although telbivudine had been reported to improve renal function,20 the high risk of antiviral drug resistance and the potential for other adverse reactions such as myopathy and polyneuropathy make this a poor choice as first - line treatment.21 tenofovir is effective in suppressing not only wild type hbv but also lamivudine, telbivudine or entecavir resistant hbv and to a slightly lesser extent adefovir resistant hbv; therefore, it is a better choice for patients who had been previously treated with other nucleos(t)ide analogues. tenofovir is also a preferred choice for women of reproductive age because of its safety record in pregnancy.22 aasld, apasl and easl guidelines recommend administration of peg - ifn for 48 - 52 weeks in both hbeag - positive and hbeag - negative patients.12,13,14 there is some variation in recommendations when nucleos(t)ide analogues can be stopped. all guidelines recommend that in hbeag - positive patients, nucleos(t)ide analogues can be stopped when the patient has completed 6 - 12 months consolidation therapy after hbeag seroconversion. the easl guidelines recommend continuing treatment until hbsag loss in patients with advanced fibrosis or cirrhosis to avoid flares associated with viral relapse. the aasld and easl guidelines recommend that in hbeag - negative patients, nucleos(t)ide analogues should be continued until hbsag loss but the apasl guidelines stated that treatment may be withdrawn after completing at least two years treatment with undetectable hbv dna documented on three occasions six months apart. all three guidelines recommend lifelong nucleos(t)ide analogue treatment in patients with cirrhosis before treatment unless they had compensated cirrhosis and had cleared hbsag. in addition to pre - treatment factors, several studies have found decrease in hbsag level after 12 or 24 weeks of peg - ifn treatment to be better predictors of response. specifically, a lack of or a small decline in hbsag level after week 12 or 24 of peg - ifn is associated with 85 - 95% negative predictive value of a response.23 these data suggest that a response - guided stop - rule may be used making peg - ifn therapy more attractive because patients who are unlikely to benefit can be spared from further side effects and costs. a recent cost - effective study showed that response - guided peg - ifn therapy is the most cost - effective approach for hbeag - positive but not hbeag - negative patients.24 thus, hbeag - positive patients with no contraindications, particularly those with genotype a hbv, high alt and low hbv dna levels before treatment who are already predicted to have a high likelihood of response to peg - ifn may be further encouraged to have a trial of peg - ifn, treatment can be stopped after 12 weeks if decline in hbsag is inadequate. a caveat to this approach is that it has not been validated prospectively and different stop rules have been proposed by different authors and for different hbv genotypes. for patients receiving nucleos(t)ide analogue therapy, the likelihood of achieving hbeag seroconversion is 40 - 50% and the likelihood of achieving hbsag loss is 0 - 10% after 5 years of continuous treatment.3 thus, more than half of the hbeag - positive patients and 95% of hbeag - negative patients will require more than 5 years of treatment. a few studies found that while viral relapse (redetection of hbv dna in serum) occurs in all hbeag - negative patients who completed 2 - 5 years treatment and who stopped treatment before hbsag loss, clinical relapse with elevated alt and hbv dna levels > 2,000 iu / ml occurred in only 50% of patients.25,26 these data suggest that hbeag - negative patients who have completed > 2 years treatment may consider stopping treatment if they are unwilling to continue or unable to afford continued treatment. however, this personalized approach must be accompanied by a plan for close monitoring for at least 6 months after discontinuation of treatment such that treatment can be promptly resumed if necessary. personalized approach to treatment of hepatitis b should adapt practice guidelines to individual patient's disease characteristics, personal preferences, medical comorbidities and social circumstances to determine when treatment should be started, which drug to use, and when to stop treatment, such that maximum benefit can be derived while minimizing costs and adverse reactions (table 3).

there are seven approved drugs for treatment of hepatitis b. professional guidelines provide a framework for managing patients but these guidelines should be interpreted in the context of the individual patient's clinical and social circumstances. personalized management of hepatitis b can be applied based on prediction of the individual patient's risk of cirrhosis and hepatocellular carcinoma to guide the frequency and intensity of monitoring and urgency of treatment. it can also be applied to decisions regarding when to start treatment, which drug to use, and when to stop based on the individual patient's disease characteristics, preference, comorbidities and other mitigating circumstances.

congenital pouch colon (cpc) is an unusual abnormality in which a pouch - like dilatation of a shortened colon is associated with an anorectal malformation (arm). this report describes a girl with type - iii cpc and uterus didelphys with a septate vagina. at puberty, the child had primary amenorrhea that was found to be due to bilateral atresia of the cervix uteri. similar findings in a girl with cpc have not been reported earlier in the literature. a 3-month - old girl was brought with the absence of the anal opening and passage of urine and stools from a single perineal opening. on examination , there was a single, fairly wide, perineal opening, and a clinical diagnosis of a persistent cloaca was made. laparotomy revealed a type - iii cpc malformation with 8 cm of normal colon proximal to the distended colonic pouch that appeared to end in a colocloacal fistula. the child had uterus didelphys with one fallopian tube and uterus flanking the distal portion of the colonic pouch and the colocloacal fistula on each side. the fistula was ligated, subtotal excision of the colonic pouch with tubularization of its outer portion performed (tubular colorraphy) and an end - colostomy fashioned. initial dissection by the posterior sagittal approach revealed a short but wide common channel (around 2 cm long) with a double vagina. the anterolateral walls of the common channel were tubularized to reconstruct the urethra over an 8 fr. catheter. with the child in the supine - lithotomy position, a short segment of ileum was isolated, its upper end sutured to the margins of the double vagina and its lower end brought to the perineum. the colostomy was mobilized and brought down to the site of the neoanus. the girl was lost to follow - up for 5 years and brought again with complaints of urinary and fecal incontinence. the child had partial urinary incontinence (ui) with passage of urine in a stream a few times daily along with continuous dribbling of urine. on examination, the urethral and vaginal openings were separate and the vaginal opening was adequate in caliber. a micturating cystourethrogram (mcu) showed a bladder of adequate capacity with a patulous bladder neck. the parents were not willing for clean intermittent catheterization or any further surgical management for ui. at 11 years of age, the girl presented with primary amenorrhea and severe monthly cyclical pain in both flanks and the pelvis. investigations were advised but the child was lost to follow - up for 3 years and brought again only when the symptoms had worsened considerably. a us of the abdomen and pelvis showed well - defined lobulated masses with cystic lesions in the pelvis and both adnexal regions. magnetic resonance imaging (mri) scan of the abdomen and pelvis showed 2 uteri and fallopian tubes that were distended and filled with blood and blood products. poorly developed cervical tissue was seen and could be traced only to the right uterus. mri scan of the pelvis showing the enlarged, distended uterine horns (right more than left) and adnexal structures (arrow) eua with cystourethroscopy showed a separate urethral orifice leading to a short urethra with a widely open bladder neck. per - vaginal examination showed tense, hard masses on both sides in the pelvis. laparotomy revealed dense pelvic adhesions, uterus didelphys with both uteruses distended with blood and blood products, bilateral endometrial cysts, and a large left hematosalpinx. the endometrial cysts were aspirated, the walls of the cysts were enucleated, and bilateral hysterectomy with left salpingectomy performed. there was very poorly developed cervical tissue without a lumen on the right side and complete cervical atresia on the left side. sections from one of the uterine horns showed endometriosis while those from the other horn were unremarkable. photograph taken during surgery showing the distended uterine horns and the left fallopian tube and adnexal structures filled with blood and hemorrhagic cysts presently, at the age of 16 years, the girl is relieved from her complaints of cyclical abdominal and pelvic pain. she is able to hold urine during the day for periods ranging from to 2 h but has severe nocturnal ui. the child is dry after an enema for 4 - 5 h. the child is fairly well - adjusted psychologically. although our study suggested that the anomalous clinical anatomy of cpc in girls as described by us was almost invariable, apart from a few exceptions, other reports, including earlier ones from our center, have not consistently described the abnormal findings of the mullerian structures. a likely reason is that in newborns and small infants, the anatomy of the external genitalia and perineum is not clear on clinical examination and endoscopic examination of the genitourinary tract is usually not feasible so that, as initially in our patient, the usual diagnosis would be that of a persistent cloaca. the vast majority of reports, including large series, have recorded only the findings at the time of primary surgery, usually in the newborn, and not the of any detailed examination, endoscopy, and/or investigations performed later. the details of reconstruction of the lower genitourinary tract have also usually not been described. there are very few reports of the obstetric implications of a double uterus and vagina (uterus didelphys) a finding that appears to be invariable in girls with types i - iii cpc. however, a report suggested that 18% of patients with uterus didelphys had an obstructed hemivagina with hematocolpos. in a recent review on the subject of gynecologic concerns in girls with arm, breech emphasized the role of vaginoscopy before puberty, preferably at the time of definitive repair of the arm. vaginoscopy allows evaluation of the vaginal anatomy and can also document the appearance, development, and position of the cervices in the vaginas and the presence or absence of mucus at the ectocervix (to infer patency). antegrade / retrograde perturbation of the mllerian structures during any open or laparoscopic procedure for the arm can confirm patency of the outflow tract. any underdevelopment of the mllerian structures can be detected by serial us starting soon after the onset of breast development. if neglected, as in our case, onset of menarche in the presence of obstruction to the outflow tract may in endometriosis, hematometra and/or hematocolpos, hematosalpinx, adnexal cysts, and chronic abdominal pain. although in the past, hysterectomy was the treatment of choice for cervical atresia, current recommendations are suppression of menses with preservation of the uterus for pregnancy with reproductive assistance, or uterovaginal anastomosis. a report of 18 cases of uterovaginal anastomosis in patients with cervical atresia reported six spontaneous pregnancies in four patients, and only one required cervical cerclage. , managed a patient of vestibular fistula with the absence of vagina and cervical atresia by anastomosing a bowel graft directly to the uterus. in , our report emphasizes the need for a comprehensive evaluation and long - term management strategy for associated gynecologic anomalies in girls with cpc, especially with regard to the patency of the outflow tract. uterus didelphys with a septate vagina appears to be invariable in girls with types i - iii cpc and needs to be assessed and managed appropriately with awareness of the possibility of obstetric complications in later life. patency of the outflow tracts should be assessed at an early stage and serial us starting soon after breast development can assess the growth of both uteruses and any evidence of obstruction to the outflow tract so that early corrective treatment can be instituted.

this report describes a girl with congenital pouch colon (cpc), uterus didelphys with septate vagina, and a cloacal anomaly. the girl underwent cloacal reconstruction at the age of 15 months. subsequently, at puberty, the child had primary amenorrhea with severe cyclic abdominal pain due to endometriosis of both the uteruses and adnexal cysts with hematometra and hematosalpinx. laparotomy with removal of both uteri and the left fallopian tube was performed. both uteri had atresia of the cervix uteri. this report emphasizes the need for comprehensive evaluation and a long - term management strategy for associated gynecologic anomalies in girls with cpc, especially with regard to patency of the outflow tract.

solid pseudopapillary tumor (spt) of the pancreas has been variously designated as solid and cystic tumors, solid and papillary epithelial neoplasms, papillary cystic neoplasms, papillary cystic tumors, and frantz's tumors. the world health organization renamed this tumor as spt in the international histological classification of tumors. spt represents a rare tumor that occurs most frequently in young women (90%). although spt is considered an indolent lesion with a low malignant potential and a favorable prognosis after surgical resection, some cases of locally infiltrating and metastatic varieties, or recurrences after surgery, have been reported. a 74-year - old woman was admitted to the hospital for fever and general weakness. on the computed tomography scan , there was large abscess cavity showing partially septated cystic lesion with peripheral inflammatory hyperemia in the right hepatic lobe. incidentally, a lobulated, 5 3.2 cm, heterogeneous pancreatic mass containing several intratumoral calcification and cystic change on the tail of the pancreas and a 6-cm, well - defined mass with heterogeneous contrast enhancement in the left lobe of the liver were detected (fig . 1). two months after conservative treatment for hepatic abscess, the abscess was completely resolved. however, the mass in the left lobe of the liver remained unchanged and then ultrasound - guided liver biopsy was performed. the tumor consisted of papillary neoplasm showing monomorphic polyhedral cells with hyalinized fibrovascular stalks, which was best classified as metastatic spt of the pancreas. immunohistochemistry was positive for vimentin, antitrypsin, and neuron specific enolase. on the presumption of an spt of the pancreas with hepatic metastasis, the patient underwent radical antegrade modular pancreatosplenectomy with hepatic resection. on operation field encapsulated masses, 6 5 cm and 5 4.5 cm in size, were found in the segment of the left liver and in the tail of the pancreas, respectively. microscopically, the growth pattern of the pancreas tumor was heterogeneous, with a combination of solid and pseudocystic structures in varying proportions. the tumor was composed of monomorphic polyhedral cells with hyalinized fibrovascular cores, thereby leading to the characteristic pseudopapillary appearance (fig . . however, perineural invasion and infiltration into the peripancreatic fat tissue could be seen . the microscopic findings of the hepatic tumor were similar to the findings of the pancreatic tumor . a final diagnosis of spt of the pancreas with metastasis to the liver was made . spt is a rare neoplasm of the pancreas accounting for only 1 to 2% of all exocrine pancreatic tumors . although spt occurs in young females and is generally considered to be low malignant potential, local recurrence or distant metastases can be found in a significant number of patients . 14.7% ( 43 of 292) were evaluated as malignant due to metastasis (22 of hepatic, lymph node metastasis or peritoneal dissemination) or invasion into adjacent organs. the prognosis for spts is excellent after curative resection (more than 90% survival at 5 years). metastasis develops in less than 15% of cases and among them hepatic metastasis is most common. suggested that complete resection was associated with long - term survival even in the presence of metastatic disease. in case report series, even patients with local recurrence as well as liver and peritoneal metastasis have experienced long - term survival. one study suggested that patient age tended to be older and the tumor size tended to be larger in metastasizing spt than in non - metastasizing spt. invasion of blood vessels, perineural clefts and adjacent organs, a high degree of cellular pleomorphism and an elevated mitotic rate are considered to associated with metastasis. nishihara et al. compared the histological features of metastasizing spt and non - metastasizing spt and reported that venous invasion, nuclear grade, and prominent necrobiotic nests are useful as histologic indicators for the malignant potential of spt. the present patient was very old age and had an incidental detection of the disease. microscopic findings showed focally - infiltrative and perineural invasion, while without lymph node metastasis and less than 5% of ki-67 positivity. in , we report a case of pancreatic spt with synchronous hepatic metastasis, which appeared later in life. preoperative diagnostic work - up is a cornerstone for surgical approach and complete resection should be considered for this potentially curative disease if operation is possible. the literatures reported up to date, as well as our case, demonstrate an aggressive approach to this rare low - grade malignant tumor can in long - term survival even in patients with local invasion and distant metastasis.

solid pseudopapillary tumor of the pancreas is a rare tumor that affects young females with low malignant potential and good prognosis with more than 90% survival at 5 years. metastasis is very rare. we report the case of a 74-year - old female who had pancreatic solid - pseudopapillary tumor and synchronous hepatic metastasis.

the choroid plays several important functions within the eye, providing metabolic support to retinal pigment epithelium (rpe) and outer retina, contributing to the blood vascularization of the preliminary portion of the optic nerve and absorbing excess of light entering the retina and rpe thanks to the presence of melanocytes. the choroid can not be excluded in the evaluation and understanding of the pathophysiological mechanisms responsible for diseases of the posterior segment. many experiments and clinical studies have shown both retinal and choroidal altered blood flow in retinitis pigmentosa (rp). clinically, the retinal vessels reduction is a pathognomonic finding in rp and the value of retinal blood flow measurement is significantly reduced in rp patients compared to that in healthy subjects. similarly in patients with rp, the ocular pulse amplitude (opa), an indirect measure of choroidal perfusion, was significantly reduced in the advanced stages of the disease. however, the role that these perfusion abnormalities have in the pathogenesis of rp is still uncertain. the recent development of the technique enhanced depth imaging (edi) , which allows through the use of spectral domain oct obtaining detailed images of the choroid, has given substantial impetus to the study of the physiopathological role of choroid such as physiological variation with age and implication in pathogenic mechanisms. in rp, the analysis of choroidal thickness has a double importance; while it is an important additional factor for the understanding of the pathogenesis and course of the disease, it is an essential element in the evaluation of therapeutic perspectives, like retinal implants, gene therapy, and stem cells therapy. retinitis pigmentosa is also a term that embraces a multitude of clinical conditions that have similar characteristics but differ for age of onset, course, and severity. the aim of the study is to portray the most common syndromic form of rp, usher syndrome type 2, analyzing the thickness of the choroid in correlation with disease stage and comparing values reported in literature on nonsyndromic rp and healthy subjects. all patients were recruited from the hereditary retinal diseases service at the university eye clinic of san paolo hospital in milan between january and may 2014. they are part of a larger group of usher syndrome patients being evaluated at our clinic. diagnosis of usher syndrome was confirmed by both clinical signs (characteristic bone spicule pigmentation, optic disc pallor, retinal vessel attenuation, visual field constriction, flat electroretinographic waves, photoreceptor atrophy shown by oct, and bilateral sensorineural hearing loss) and of genetic analysis. we considered for the present study patients with at least one homozygous mutation or two heterozygous mutations. heterozygous mutations have been confirmed on the two alleles by segregation analysis (table 1). all participants underwent a complete ophthalmologic examination including best corrected visual acuity (bcva), intraocular pressure, axial length, automated visual field, and edi oct. demographic data and medical history (including the age of diagnosis of usher syndrome, the first symptoms onset, and the presence / absence of other members of the family affected by the same disease) were collected using a questionnaire that all the patients were asked to complete before the inclusion in the study. factors that have been demonstrated to influence choroidal thickness (refractive error > 6 diopters and elongated axial length) or poor oct scan quality (media opacities, nystagmus) were considered noninclusion criteria. other exclusions included history of glaucoma or uveitis, prior ocular surgery (excepted cataract surgery), systemic cardiovascular disease like hypertension, and diabetes. we arbitrarily considered 3 stages of disease according to the parameters of visual acuity and visual field: stage 1 for patients with visual acuity better than 0.6 decimal or visual field mean defect less than 10 db, stage 2 for patients with visual acuity from 0.6 to 0.2 decimal or visual field mean defect from 10 to 20 db, and stage 3 for patients with visual acuity less than 0.2 decimal or visual field mean defect more than 20 db. visual field analysis was obtained using the 30 - 2 sita standard program of the humphrey visual field analyzer (zeiss / humphrey systems, dublin, ca, united states). examinations were performed using the best correction for near vision. for the purpose of the study mean defect (md) and pattern standard deviation (psd) values were considered. axial length was measured using the iol master 500 biometer (carl zeiss meditec, dublin, ca, united states). the values used for the study were obtained from the mean of five consecutive measures. retinal and choroidal imaging were obtained using spectralis hra and oct (heidelberg engineering, heidelberg, germany). edi oct technique, previously described by spaide and margolis , allows detailed imaging of the choroidal layer. for the study we took single line scans of 30 composed of 100 averaged images using the automatic eye tracking software across the fovea both vertically and horizontally. both retinal and choroidal measures were taken manually in a masked fashion by two experienced oct - readers as shown in figure 1.: from the inner border of the sclera to the outer border of the rpe vertically using the calipers of the heidelberg reader software subfoveally and at 500 mm intervals for 2.5 mm nasal, temporal, superior, and inferior to the centre of the fovea. retinal thickness was taken in the same intervals vertically from the outer border of the rpe to the inner border of ilm. horizontal spatial distribution of photoreceptor layer was manually measured using the calipers. also presence / absence of cystoid macular edema was considered. only oct scans of good quality were used for the measures (figure 1). first, we studied the correlation between choroidal thickness and retinal thickness in the foveal region with a series of predictors (age, time from first symptoms, axial length, bcva, md, and psd as continuous variables, presence of photoreceptors, and presence of oedema as categorical variables). all correlations were calculated using univariate mem models where a patient random effect was included to account for the covariance among observations from the same subject (i.e., two measurements from each patient, one for each eye). secondly, we analyzed the effect of the age of the patients on the choroidal thickness at various distances from the fovea (every 0.5 microns from 2.5 to 2.5 microns away) on both temporal - nasal and superior - inferior axes. in this case age and the site of measurement were treated as fixed effects; a patient and an eye random effect were used to correct for the covariance among observations from the same patient and the same eye (i.e., multiple measurements from each eye of each patient). finally, an interaction term was included to allow different slopes in the model at each site. the different slopes were used to create a correlation map showing the effect of the age of the patient on the choroidal thickness at each site. z - test analysis was used to compare our data on choroidal thickness in patients with usher syndrome type 2 with values of choroidal thickness in healthy and nonsyndromic rp patients as reported in published literature. 10 patients (20 eyes) with clinical and genetic diagnosis of usher syndrome type 2 were included in the study (4 men and 6 women). the mean age was 43.1 13.09 years; the mean duration of disease was 17.8 12.28 years. spherical equivalent ranged from 3.5 d to + 2.5 d and mean axial length was 23.59 1.29 mm (table 2). mean subfoveal choroidal thickness (sfct) (table 3) was 237.45 76.81 microns, with a mean choroidal thickness thinner 2.5 mm nasally than 2.5 mm temporally to the fovea (130.2 and 194.6 mm, resp .) and 2.5 mm inferiorly than 2.5 mm superiorly to the fovea (200.1 and 219.75 mm, resp .). the mean retinal thickness was thinner 2.5 mm temporally than 2.5 mm nasally to the fovea (199.9 and 223.85 mm, resp .) and 2.5 mm inferiorly than 2.5 mm superiorly to the fovea (209.35 and 222.55 mm, resp .). 25% of the eyes (5/20) presented cme. in 15% of the eyes (3/20) photoreceptors outer segment layer was not detectable at oct. when detectable, the mean horizontal extension of photoreceptor layer (concentric to the fovea) was 264.7 148.71 microns. sfct was significantly correlated with age: sfct decreases with age (estimated coefficient 4.412, pearson correlation coefficient 0.725, p < 0.01). no statistically significant correlation was found between sfct and duration of disease, different stages of disease (as evaluated with visual acuity and visual field parameters), retinal thickness, and axial length (table 4). we then analyzed the correlation between choroidal thickness and age in each interval: figure 2 shows the intervals in which the correlation was significantly different from that of the fovea. the intervals further away from the fovea were less correlated with age (the values indicated with the asterisks). choroidal thickness values from usher patients were compared with values reported in published literature for healthy subjects and nonsyndromic rp patients. choroidal thickness in usher patients was significantly reduced when compared to healthy subjects (p < 0.01, z - test), while no difference was found when compared to choroidal thickness from nonsyndromic rp patients (p = 0.72). our case control study showed that choroidal thickness is reduced in people with usher syndrome type 2 if compared to healthy subjects. our confirmed those reported by dhoot et al., who studied choroidal thickness in nonsyndromic rp using the same strategy to analyze oct images, by ayton et al. who studied nonsyndromic rp patients using a graphic software to analyze choroidal thickness in oct scans and by yoon and yu who used a different instrument of acquisition and analysis of oct scans. both retinal vascularization and choroidal vascularization were reported to be significantly reduced in rp patients and in animal models of rp: our observations can be considered a sign of this blood flow reduction. recent studies demonstrated different visual acuity, visual field, and cone electroretinogram amplitude loss in usher syndrome patients if compared to nonsyndromic type of rp: comparison between our data and choroidal thickness values in nonsyndromic rp reported in literature could not explain these differences. although previous studies reported that choroidal arteries and choriocapillaris under macular region fill more rapidly and intensely compared to other retinal locations, supporting the concept that thicker choroidal thickness under the fovea is due to macular higher metabolic request; however, similarly to yeoh et al. and dhoot et al., our study did not show any correlation between sfct and visual acuity. we also could not find any correlation between sfct and presence / absence of outer retinal layer or macular edema. a possible explanation of these data could be that edi oct technique does not allow distinguishing choriocapillaris, which represents about 10% of all choroidal thickness , from the rest of choroidal volume. first publications by spaide and margolis on choroidal thickness in healthy subjects reported a decrease of choroidal thickness with age. similarly our study found a statistically significant correlation: also in patients with usher syndrome type 2 sfct decreases with age. on the other hand, no correlation between sfct and duration of disease was found, in contrast with ayton's previous report. this observation could be due to the fact that it is difficult to establish the exact onset of the disease: for the majority of patients diagnosis follows symptoms onset and in addition to that considering that usher syndrome is a genetic disease the concept of duration of disease is not as relevant as chronological age. interestingly in our usher syndrome study group the topographic choroidal thickness variation appears to be similar to topographic choroidal thickness variation previously reported in literature for healthy subject. choroidal thickness was thinner in nasal than in temporal sector and in inferior than superior sector. furthermore we evaluated the correlation between choroidal thickness and age in each topographic sector: as shown in figure 2 intervals far from the fovea ed in being statistically less associated with age if compared to the fovea. this fact could be reasonably explained by the fact that central retina, particularly the fovea, is the last involved with the degenerative process in rod - cone dystrophy. to our knowledge, the relationship between the thickness of the choroid in different regions of the macula and the correlation with topographic variations has not yet been described. lack of correlation between choroidal thickness and axial length could be explained by the small number of patients studied and by the fact that we considered an exclusion criteria refractive error > 6 diopters. in we have demonstrated in vivo choroidal thickness reduction in different topographic sectors in patients with usher syndrome type 2. these data are important not only for the comprehension of pathogenetic mechanisms in hereditary retinal diseases but also for the evaluation of therapeutic approaches. a deep knowledge of residual choroidal blood flow and changes in choroidal vascularization would be necessary for both retinal implant and stems cells therapy. limits of our study are represented by the small number of patients and the absence of a healthy and a nonsyndromic rp control group. further studies will need to investigate the correlation between different type of rp, classified by genetic mutation, and choroidal changes. new and more sophisticated oct techniques will allow better understanding of the role that the choroid plays in the pathogenesis of hereditary retinal diseases.

to portray usher syndrome type 2, analyzing choroidal thickness and comparing data reported in published literature on rp and healthy subjects. methods. 20 eyes of 10 patients with clinical signs and genetic diagnosis of usher syndrome type 2. each patient underwent a complete ophthalmologic examination including best corrected visual acuity (bcva), intraocular pressure (iop), axial length (al), automated visual field (vf), and edi oct. both retinal and choroidal measures were measured. statistical analysis was performed to correlate choroidal thickness with age, bcva, iop, al, vf, and rt. comparison with data about healthy people and nonsyndromic rp patients was performed. . mean subfoveal choroidal thickness (sfct) was 248.21 79.88 microns. sfct was statistically significant correlated with age (correlation coefficient 0.7248179, p < 0.01). no statistically significant correlation was found between sfct and bcva, iop, al, vf, and rt. sfct was reduced if compared to healthy subjects (p < 0.01). no difference was found when compared to choroidal thickness from nonsyndromic rp patients (p = 0.2138). . our study demonstrated in vivo choroidal thickness reduction in patients with usher syndrome type 2. these data are important for the comprehension of mechanisms of disease and for the evaluation of therapeutic approaches.

nursing education aims to provide an appropriate level of knowledge and skills in nursing students. improving the quality of nursing care requires obtaining high levels of knowledge and skills during nursing training period. however, skills acquisition is a complex process and needs students to acquire a combination of psychomotor, cognitive and affective skills and incorporate such skills with their theoretical knowledge, procedural skills and critical thinking. several studies in different countries reported that nursing education does not always prepare its students for the demands of working as a professional registered nurse. newly graduated nurses may have weak practical skills, which can be a threat to patients safety. a meta - analysis concluded that clinical skills training in nursing had not led to deep learning. investigated student nurses experience of learning in clinical environments and reported similar findings. shah et al. studied the process of motor skill acquisition and reported that most students complain about their problems in transferring what they learned at clinical skills centers to the real clinical setting. several factors are involved, among them education methods play a crucial role. therefore, it is imperative for nurse trainers to use the best methods to promote learning and skill acquisition in their students. concept mapping is a modern educational strategy based on the ausubel s learning assimilating theory. according to ausubel, meaningful learning occurs when newly learned materials can be linked to the information previously stored in the brain. novak and govin defined concept mapping as a graphical method for presenting a set of concepts placed in a thematic framework. in this perspective, we think and learn the concepts by linking new concepts to previously known concepts in a systematic manner. in fact, concept mapping is an active teaching method, which helps nurse educators to develop critical thinking and problem solving capabilities in graduates. several studies in the field of medical education used concept mapping in teaching theoretical courses, teaching and evaluation of creativity skills, teaching students to develop and record care plans. however, most studies used grades and students academic achievement to measure the effect of concept mapping. moreover, some studies had weaknesses in their design and methodologies. adlaon compared the effect of a three - week teaching course using concept mapping and traditional teaching methods on students performance in responding multiple choice questions and reported that concept mapping was superior to traditional teaching method. however, the short duration of intervention, the small sample size, the small numbers of concept maps used and the type of evaluation made the difficult to be generalized. in another study, reported that contradictory of concept mapping technique might be attributed to small sample size, inappropriate gauges, non - random selection and lack of control group in different studies. most studies assessed the effects of concept mapping on students cognitive learning domain and the effect of this method on students skill acquisition had not been assessed so far. then, we can not generalize the of these studies to the psychomotor domain. this study was performed to compare the effects of training using concept mapping and traditional teaching methods on practical skills of nursing students. this quasi - experimental pretest and post - test study was conducted on 70 students enrolled for the first time in a practical course on fundamentals of nursing. the study was conducted in the second semester of the academic year 2013 - 2014 in the clinical skills center at tehran university of medical sciences (tums). the inclusion criterion was having no previous experience on concept mapping and direct observation of procedural skills (dops) methods. an absence of more than three sessions from the training sessions was the exclusion criterion. the sample size was calculated using the of a previous study by karimi - moneghi et al. in which s1, s2, 1 and 2 were respectively 1.5, 1, 16.2, and 15.25. then, with a type i error of 0.05 and a power of 0.80, the sample size was estimated as 29 students for each group. however, we recruited 35 students with inclusion criteria in each group to compensate for possible attritions. then, among a total of 140 students, 70 students with inclusion criteria who consented to participate in the study were recruited. then, the students were randomly assigned in the intervention (n = 35) or the control group (n = 35) using a random numbers table (figure 1). before commencing the intervention, all the students were invited to the clinical skills center. then, a pre - test was performed in both groups using the dops method. afterward, each group was though 14 sessions of 45 minutes, two sessions per week, through either concept mapping (intervention group) or the traditional method (control group). a two - part instrument was used to collect data in this study including a demographic data form and a checklist for assessing the students skills in selected procedures. the demographic data form consisted of questions regarding students age, gender, grade point average (gpa), living location, marital status and the group in which the student was assigned to. the checklist used to observe student s skills in selected practical procedures consists of 9 items regarding having comprehensive knowledge of the indications and the relevant anatomy, obtaining patient s consent for the procedures, implementing all required preparations before the procedures, implementing the aseptic techniques, the technical ability to perform the procedure, implementing the required nursing care after the procedures, appropriate using of communication skills, using essential ethical and professional conduct and the observer s overall opinion on student s performance. all items scored on a 10-point likert scale ranged from zero to 9, totaling a score of 0 to 81 for each skill. farsi version of this instrument was also validated by eleven faculty members in zahedan nursing and midwifery college and presented appropriate content validity. its reliability was also checked through internal consistency method and the cronbach s alpha was 0.94. the intervention group was trained using concept mapping method followed by demonstrating relevant skills by instructors and students practices. all maps were spider shaped so that the main concepts were in the middle of maps and sub - concepts were presented in the sides. in all maps, the teacher presented the material in a clockwise direction from the starting point. at the first session , the intervention group was informed that special software would be used to teach them in this course. after each session, every student was required to design a concept map of the whole proposed lecture for the next session individually; then, at the beginning of the next session, a few concept maps prepared by the students were reviewed and feedback was given to them. all training sessions of the intervention group were held on saturdays and sundays, at the clinical skills center of nursing school of tums. in this study, the instructor (the second researcher drew concept maps using offline method in free - mind ( gnu general public license, java), based on the literature on websites and specialized instructions. the control group was also trained in 14 sessions using conventional method (i.e. lecturing, demonstration and then students practicing). the training sessions of this group were held on different days (tuesdays and wednesdays) to prevent exchanging the information about concept mapping between the two groups. the curriculum of both groups was the same and the only difference was used teaching methods. the skills taught included intradermal injection, subcutaneous injection, intramuscular injection, intravenous cannulation, intravenous injection, intravenous fluids preparation, nasogastric catheterization, feeding through a nasogastric catheter (gavage), nasogastric lavage, oxygen therapy methods (through nasal cannula and face mask), measuring the blood pressure, airway suctioning, tracheostomy care and dressing change. the list of procedures was confirmed by the authorities in nursing school and the content validity of all concept maps were confirmed by 10 experienced nursing faculty members in the nursing school of tums. four weeks after the last training session, dops functional test was conducted without previously informing in the clinical skills center for all students in the both groups. two lecturers of the fundamentals of nursing course in nursing and midwifery school at tehran university of medical sciences (who were trained on dops and how to observe the students using special checklists, but did not know anything about the project), conducted all testing procedures. before starting the testing , all the students were gathered in a classroom and briefed about the dops and what should they do during the testing session. each student was required to select three procedures (i.e. cleaning, injection and sterilizing) among several procedures already taught to them. the name of procedure was written on pieces of paper and put in three boxes. the students performed the selected procedures under direct observation of the aforementioned specialists who completed the dops checklists. finally the mean of students' scores was calculated for each individual skill and recorded at the top of the checklist. the present study was approved by the institutional review board and the research ethics committee of tums. all students were aware that they are under investigation and signed an informed consent at the beginning of study. statistical analysis was performed using spss-13 software (spss inc, chicago, il, usa). independent samples t - test was used to compare the mean of procedural skills scores, the mean age and gpa of the two groups. paired t - test was used to compare the mean scores on each group before and after the intervention. chi - square test was used to compare gender differences and fisher s exact test for marital status and residential area of the two groups. this quasi - experimental pretest and post - test study was conducted on 70 students enrolled for the first time in a practical course on fundamentals of nursing. the study was conducted in the second semester of the academic year 2013 - 2014 in the clinical skills center at tehran university of medical sciences (tums). the inclusion criterion was having no previous experience on concept mapping and direct observation of procedural skills (dops) methods. an absence of more than three sessions from the training sessions was the exclusion criterion. the sample size was calculated using the of a previous study by karimi - moneghi et al. in which s1, s2, 1 and 2 were respectively 1.5, 1, 16.2, and 15.25. then, with a type i error of 0.05 and a power of 0.80, the sample size was estimated as 29 students for each group. however, we recruited 35 students with inclusion criteria in each group to compensate for possible attritions. then, among a total of 140 students, 70 students with inclusion criteria who consented to participate in the study were recruited. then, the students were randomly assigned in the intervention (n = 35) or the control group (n = 35) using a random numbers table (figure 1). before commencing the intervention, all the students were invited to the clinical skills center. then, a pre - test was performed in both groups using the dops method. afterward, each group was though 14 sessions of 45 minutes, two sessions per week, through either concept mapping (intervention group) or the traditional method (control group). a two - part instrument was used to collect data in this study including a demographic data form and a checklist for assessing the students skills in selected procedures. the demographic data form consisted of questions regarding students age, gender, grade point average (gpa), living location, marital status and the group in which the student was assigned to. the checklist used to observe student s skills in selected practical procedures consists of 9 items regarding having comprehensive knowledge of the indications and the relevant anatomy, obtaining patient s consent for the procedures, implementing all required preparations before the procedures, implementing the aseptic techniques, the technical ability to perform the procedure, implementing the required nursing care after the procedures, appropriate using of communication skills, using essential ethical and professional conduct and the observer s overall opinion on student s performance. all items scored on a 10-point likert scale ranged from zero to 9, totaling a score of 0 to 81 for each skill. the checklist for dops farsi version of this instrument was also validated by eleven faculty members in zahedan nursing and midwifery college and presented appropriate content validity. its reliability was also checked through internal consistency method and the cronbach s alpha was 0.94. the intervention group was trained using concept mapping method followed by demonstrating relevant skills by instructors and students practices. all maps were spider shaped so that the main concepts were in the middle of maps and sub - concepts were presented in the sides. in all maps, the teacher presented the material in a clockwise direction from the starting point. at the first session , the intervention group was informed that special software would be used to teach them in this course. after each session, every student was required to design a concept map of the whole proposed lecture for the next session individually; then, at the beginning of the next session, a few concept maps prepared by the students were reviewed and feedback was given to them. all training sessions of the intervention group were held on saturdays and sundays, at the clinical skills center of nursing school of tums. in this study, the instructor (the second researcher drew concept maps using offline method in free - mind ( gnu general public license, java), based on the literature on websites and specialized instructions. the control group was also trained in 14 sessions using conventional method (i.e. lecturing, demonstration and then students practicing). the training sessions of this group were held on different days (tuesdays and wednesdays) to prevent exchanging the information about concept mapping between the two groups. the curriculum of both groups was the same and the only difference was used teaching methods. the skills taught included intradermal injection, subcutaneous injection, intramuscular injection, intravenous cannulation, intravenous injection, intravenous fluids preparation, nasogastric catheterization, feeding through a nasogastric catheter (gavage), nasogastric lavage, oxygen therapy methods (through nasal cannula and face mask), measuring the blood pressure, airway suctioning, tracheostomy care and dressing change. the list of procedures was confirmed by the authorities in nursing school and the content validity of all concept maps were confirmed by 10 experienced nursing faculty members in the nursing school of tums. four weeks after the last training session, dops functional test was conducted without previously informing in the clinical skills center for all students in the both groups. two lecturers of the fundamentals of nursing course in nursing and midwifery school at tehran university of medical sciences (who were trained on dops and how to observe the students using special checklists, but did not know anything about the project), conducted all testing procedures. before starting the testing, all the students were gathered in a classroom and briefed about the dops and what should they do during the testing session. each student was required to select three procedures (i.e. cleaning, injection and sterilizing) among several procedures already taught to them. the name of procedure was written on pieces of paper and put in three boxes. the students performed the selected procedures under direct observation of the aforementioned specialists who completed the dops checklists. finally the mean of students' scores was calculated for each individual skill and recorded at the top of the checklist. the present study was approved by the institutional review board and the research ethics committee of tums. all students were aware that they are under investigation and signed an informed consent at the beginning of study. statistical analysis was performed using spss-13 software (spss inc, chicago, il, usa). independent samples t - test was used to compare the mean of procedural skills scores, the mean age and gpa of the two groups. paired t - test was used to compare the mean scores on each group before and after the intervention. chi - square test was used to compare gender differences and fisher s exact test for marital status and residential area of the two groups. for gender distribution, 58.1% of the students in the control group and 56.9% of the intervention group were females. no significant difference was observed regarding demographic characteristics of the two groups (table 1). gpa: grade point average table 2 presents students mean scores in three skills of cleaning, injection and sterilizing before and after the education. before education , no significant differences were observed between the two groups in the three skills of cleaning (p = 0.251), injection (p = 0.185), and sterilizing (p = 0.568) (table 2). the students mean scores significantly increased after the education and the difference between pre and post intervention of the students mean scores were significant in the both groups at < 0.001. however, after education, in all three skills, the mean scores of the intervention group were significantly higher than the control group (p < 0.001 ; table 2). to the best of our knowledge, this was the first study comparing the effect of concept mapping and traditional methods of skill teaching on nursing students learning in practical skills. the study revealed that both methods could significantly increase the mean score of students at posttest. however, students posttest mean scores were significantly higher in the group taught via concept mapping method. our finding on positive effect of concept mapping on learning was in line with some previous studies such as van bon - martens, gul and boman and magnussen, harrison and gibbons, and gonzalez et al. though not on psychomotor domain, all of these studies confirmed that concept mapping is superior to conventional methods in enhancing students learning in the cognitive domain. although both teaching methods used in this study could enhance students learning, nurse teachers are responsible to find, develop and implement more effective methods on students learning. this fact would be more important in the psychomotor domain that is more pertinent to patients safety and the quality of nursing services. therefore, it is suggested to integrate concept mapping as an effective method in our conventional methods of skill teaching. inconsistent with our findings, hsu and hsieh reported that although concept mapping had some positive effects on learning, the effect was not statistically significant. however, our study and several previous investigations showed that concept mapping would be effective if appropriately implemented. concept mapping shows concepts in a hierarchical format from general to detail, such as a pyramid structure of information in long - term memory. therefore, concepts learned via this way can remain for a long - term and increase learning significantly. concept mapping also allows students to understand the errors in theirs understanding of concepts and then would increase their motives to correct such errors. concept mapping combines the problem - oriented learning, critical thinking, self - directing learning and enhances students performance in the training group. scahill et al. also reported that concept mapping improves students learning and academic achievement with a positive effect on their attitudes. when a concept map is drawn by the students, they integrate their new knowledge with what they have learned previously. this method may help students to easily classify their knowledge, make them coherent to make a deeper understanding. this would assist students reaching higher levels of cognitive learning rather than memorizing a series of concepts. this study showed that concept mapping was superior to conventional skill teaching methods. considering the of this study, it is suggested to use concept mapping in teaching practical courses such as fundamentals of nursing. to this end, nurse teachers should be trained to integrate concept mapping in their teaching and learning activities. however, the nature of each course, instructor s mastery and types of learners can affect selecting teaching method. this study was implemented in a small sample and compared the concept mapping with conventional methods. therefore, it is suggested to perform studies using concept mapping in other practical courses in nursing and to compare it with other active teaching methods. in this study, the effect of intervention was assessed in the skill center not in the real clinical setting. therefore, investigating the effect of concept mapping on students competencies in real clinical setting is suggested.

: development of practical skills in the field of nursing education has remained a serious and considerable challenge in nursing education. moreover, newly graduated nurses may have weak practical skills, which can be a threat to patients safety.objectives:the present study was conducted to compare the effect of concept mapping and conventional methods on nursing students practical skills.patients and methods: this quasi - experimental study was conducted on 70 nursing students randomly assigned into two groups of 35 people. the intervention group was taught through concept mapping method, while the control group was taught using conventional method. a two - part instrument was used including a demographic information form and a checklist for direct observation of procedural skills. descriptive statistics, chi - square, independent samples t - tests and paired t - test were used to analyze data.:before education, no significant differences were observed between the two groups in the three skills of cleaning (p = 0.251), injection (p = 0.185) and sterilizing (p = 0.568). the students mean scores were significantly increased after the education and the difference between pre and post intervention of students mean scores were significant in the both groups (p < 0.001). however, after education, in all three skills the mean scores of the intervention group were significantly higher than the control group (p < 0.001).: concept mapping was superior to conventional skill teaching methods. it is suggested to use concept mapping in teaching practical courses such as fundamentals of nursing.

retinoblastoma is the most common intraocular tumor of childhood and accounts for 11% of all cancers that occur during the first year of life. because treatments for retinoblastoma cure over 90% of patients, organ and vision preservation and the minimization of late treatment side - effects are important secondary treatment goals. retinoblastoma had been treated by external beam radiotherapy (ebr), and for many years this was the accepted treatment standard. however, greater knowledge of radiation induced morbidities and of secondary tumor risks after radiation therapy have encouraged the use of primary chemotherapy plus conservative focal therapy over the past decade. despite the recent trend toward chemoreduction, radiotherapy remains an excellent means of preserving vision in children (aged > 1 yr) with retinoblastoma, because the tumor is radiosensitive and routinely responds to radiotherapy. moreover, technologic advances made in the radiation oncology enable more precise targeting for tumor, avoiding healthy tissues, and the risks of secondary nonocular cancer reduced. brachytherapy has been used for selected cases in expert hospitals, and stereotactic conformal radiotherapy, and proton therapy could also be considered components in the modern radiotherapy armamentarium for retinoblastoma. however, available clinical data for stereotactic conformal therapy and proton therapy is limited. the korea cancer center hospital (kcch) has considerable experience of treating retinoblastoma in korea, and thus, we retrospectively reviewed our experiences of treating retinoblastoma patients with ebr, as initial treatment, to determine its long - term effects on subsequent tumor control, and its associated complication rates and prognostic factors. in addition, we reviewed ebr and brachytherapy clinical data in the hope of providing guidelines regarding indications for radiation therapy in patients with retinoblastoma. the medical records of all patients diagnosed as retinoblastoma who received ebr as an initial treatment at the kcch between july 1987 and june 1998, were reviewed. a total of 36 eyes in 29 patients with intraocular retinoblastoma underwent ebr as an initial treatment. of these, 4 patients were excluded due to a short follow - up duration (<3 yr). patient details and data concerning tumor features, treatment parameters, and complications were collected by chart review. briefly, the study subjects were 21 boys and 4 girls of median age at treatment commencement of 7.1 months (range 7 weeks to 65 months). the most frequent presenting finding was leukocoria in 16 patients (64%) followed by strabismus in 6 (24%). for the 21 patients with bilateral involvement, 28 eyes received external beam radiation, and 14 eyes were enucleated due to advanced disease without visual potential before ebr. of the 32 eyes treated, 0 were of reese - ellsworth (re) group i, 5 were group ii, 16 were group iii, 4 were group iv, and 7 were group v (5 in re group va and 2 in re group vb). treatment simulation was done for with patients under sedation while wearing a thermoplast head mask immobilization device. all patients were treated in the supine position using a linear accelerator at a photon energy of 6 mv with compensating bolus as needed. twenty patients (27 eyes) were treated using opposed lateral fields alone (mainly patients with bilateral disease), while five patients (5 eyes) were treated using anterior and lateral wedged pair fields with no attempt to shield the lens. the most frequently used field size (excluding half beam blocking) was 44 cm, but field sizes ranged from 33.5 cm to 64 cm. treatment doses ranged from 35 to 59.4 gy (median 41.6 gy) in fractions of 1.6 to 2.0 gy. chemotherapy was usually administered for high stage contralateral tumors that had been enucleated to control microscopic tumors. twenty - two patients received cyclophosphamide plus vincristine with or without doxorubicin at various doses and cycle numbers. enucleation was performed in cases with definite tumor progression after additional treatment or due to a severe complication. evaluations were performed at each follow - up to determine tumor sizes and visual acuities, and to detect new lesions and complications, such as, retinopathy, cataract, neovascular glaucoma, and midfacial hypoplasia. in addition, we analyzed prognostic factors, such as, gender, age, and the re classification according to ocular survival using the log - rank test. in addition, the relationship between re classification and visual acuity was analyzed using the t - test. overall survival was calculated from ebr commencement to final follow - up or death, whereas ocular survival was calculated from ebr commencement to time of enucleation or final follow - up with an intact eye. the medical records of all patients diagnosed as retinoblastoma who received ebr as an initial treatment at the kcch between july 1987 and june 1998, were reviewed. a total of 36 eyes in 29 patients with intraocular retinoblastoma underwent ebr as an initial treatment. of these, 4 patients were excluded due to a short follow - up duration (<3 yr). patient details and data concerning tumor features, treatment parameters, and complications were collected by chart review. briefly, the study subjects were 21 boys and 4 girls of median age at treatment commencement of 7.1 months (range 7 weeks to 65 months). the most frequent presenting finding was leukocoria in 16 patients (64%) followed by strabismus in 6 (24%). for the 21 patients with bilateral involvement, 28 eyes received external beam radiation, and 14 eyes were enucleated due to advanced disease without visual potential before ebr. of the 32 eyes treated, 0 were of reese - ellsworth (re) group i, 5 were group ii, 16 were group iii, 4 were group iv, and 7 were group v (5 in re group va and 2 in re group vb). treatment simulation was done for with patients under sedation while wearing a thermoplast head mask immobilization device. all patients were treated in the supine position using a linear accelerator at a photon energy of 6 mv with compensating bolus as needed. twenty patients (27 eyes) were treated using opposed lateral fields alone (mainly patients with bilateral disease), while five patients (5 eyes) were treated using anterior and lateral wedged pair fields with no attempt to shield the lens. the most frequently used field size (excluding half beam blocking) was 44 cm, but field sizes ranged from 33.5 cm to 64 cm. treatment doses ranged from 35 to 59.4 gy (median 41.6 gy) in fractions of 1.6 to 2.0 gy. chemotherapy was usually administered for high stage contralateral tumors that had been enucleated to control microscopic tumors. twenty - two patients received cyclophosphamide plus vincristine with or without doxorubicin at various doses and cycle numbers. enucleation was performed in cases with definite tumor progression after additional treatment or due to a severe complication. evaluations were performed at each follow - up to determine tumor sizes and visual acuities, and to detect new lesions and complications, such as, retinopathy, cataract, neovascular glaucoma, and midfacial hypoplasia. in addition, we analyzed prognostic factors, such as, gender, age, and the re classification according to ocular survival using the log - rank test. in addition, the relationship between re classification and visual acuity was analyzed using the t - test. overall survival was calculated from ebr commencement to final follow - up or death, whereas ocular survival was calculated from ebr commencement to time of enucleation or final follow - up with an intact eye. median follow up was 150 months (range 55 - 249 months), and the 10-yr ocular and overall survival rates were 75.4% and 92.3%, respectively (fig . of these, 7 eyes were enucleated and 2 were salvaged by cryotherapy and laser treatment ( table 1). one additional enucleation was performed due to phthisis bulbi (patient no . 2 in table 1). therefore, ocular preservation was achieved in 24 of the 32 (75.4%) eyes. according to re classification, 4 of 5 eyes were retained in group ii, 13 of 16 in group iii, 2 of 4 in group iv, and 5 of 7 in group v. vision was preserved in 24 (75%) out of 32 treated eyes. of the preserved 24 eyes, 9 (37.5%) had a visual acuity better than 20/40; 5 (20.8%) had an acuity worse than 20/40 but better than 20/200, and 10 (41.7%) had vision worse than 20/200. all the patients with visual acuity less than 20/200 presented with the involvement of the posterior pole, and showed macular degeneration following treatment. records showed that 11 patients experienced a radiation - induced complication; 6 had cataracts alone, 1 had retinal detachment alone and 4 had one more complications (table 1). nine cataracts were recorded and removed when necessary, and vision restoration or improvement was achieved in all cases. the median time to cataract development was 5 yr and 9 months (range 13 to 125 months). serous retinal detachment was detected in 3 eyes; 2 were transient and reattached after 3 months and 6 months respectively. an age of less than 13 months was found to be a significantly favorable factor of ocular survival by univariate analysis, but this was not confirmed by multivariate analysis. no other prognostic factor was identified during this study (table 2), and in particular, no significant relationship was found between re classification and visual acuity. ebr has a valuable role in the treatment of retinoblastoma, but radiation - induced secondary tumors jeopardize the role played by ebr in retinoblastoma. large - scale cohort studies performed to quantify cancer risks in retinoblastoma treated by radiotherapy have found that radiotherapy contributed significantly to the risks of developing brain, nasal cavity, and eye and orbit cancers. notably, the risk of cancer of the nasal cavity increased by 1,364-fold in hereditary retinoblastoma treated by ebr. external beam radiation is usually favored for the treatment of bilateral retinoblastoma, which is almost always hereditary, and thus, all possible efforts should be made to reduce the risk of secondary malignancies after radiotherapy. a review of ebr technology revealed that the lateral field is usually used because it requires lower lens doses. in most cases, however, reductions in doses administered to the orbital cavity, optic nerve, or cranial bone appear to be more important. recently, more meticulous techniques, such as, intensity modulated radiation therapy (imrt) and stereotactic hypofractionated radiation therapy, have been introduced, which administer lower doses of radiation to critical organs. reisner et al. compared several ebr techniques, that is, electron beam, the lateral 2 field and anterior - lateral 2 field techniques, and imrt, and found that imrt had an advantage over the other techniques, because it allowed greater dose reductions to the orbit and lacrimal gland, while maintaining therapeutic doses to the ora serrata retinae and vitreous. recently, sahgal et al. reported that stereotactic fractionated radiation therapy for localized tumor masses can achieve markedly lower doses to surrounding critical normal tissues than conventional radiation therapy. furthermore, proton therapy is also likely to reduce cranial bone radiation dose due to the radiation quality of the bragg peak. brachytherapy was introduced in the 1920's to treat ocular tumors and reduce the exposure to normal tissue around tumors, and has been further developed in terms of new radioisotopes, implant designs, and techniques of placement. table 3 details the ocular survival and cataract incidence rates of conventional radiation therapy techniques and brachytherapy; it is worth noting that the radiation doses, fraction numbers, and beam delivery techniques used at different institutes over the last 20 yr has varied considerably. the literature review revealed that ocular survival ranged from 38% to 84% (median 73%) for ebr, but from 60% to 95% (median 79%) for brachytherapy. furthermore, radiation - induced cataract was found to be the most common complication of radiation therapy; the incidence of cataract ranged from 22% to 41% (median 28%) for ebr and from 10% to 43% (median 17%) for brachytherapy. however, it should be noted that because retinoblastoma is rarely encountered in infants, skilled treatment is required to obtain good outcomes after ebr and brachytherapy in the present study, we analyzed patient and ocular survivals, and long - term treatment toxicities. our , in terms of ocular survival and the incidence of cataract, compare well with those of other ebr series. long - term follow - up findings revealed that late radiation effects were milder than expected, and that favorable visual outcome had been maintained. furthermore, ocular survival analysis by re classification, despite the limitations imposed by small patient numbers, showed that involved eyes with an re classification of 4 or 5 achieved 64% ocular survival, which indicates that radiation therapy should be considered before enucleation as primary or secondly treatment for even advanced retinoblastoma. in , our long - term show that ebr has an important role to play in the avoidance of enucleation in retinoblastoma with small lesions or advanced lesions with vitreous seeding. furthermore, non - conventional radiation therapies as brachytherapy, stereotactic radiotherapy, imrt, and proton therapy, are likely to reduce complication rates. additional research is required to establish new indications for the various radiation therapy techniques available for the treatment of retinoblastoma.

the authors reviewed their experiences of external beam radiotherapy (ebr) as an initial treatment in retinoblastoma patients to determine its long - term effect on subsequent tumor control and complications. a total of 32 eyes in 25 patients that underwent ebr for retinoblastoma were reviewed retrospectively. the patients consisted of 21 boys and 4 girls of median age at treatment of 7.1 months. radiation doses ranged from 35 to 59.4 gy. the 10-yr ocular and patient survivals were 75.4% and 92.3%, respectively. nine of the 32 eyes progressed; 7 of these were enucleated and 2 were salvaged by focal treatment. according to the reese - ellsworth classification, 4 of 5 eyes of group ii, 13 of 16 group iii eyes, 2 of 4 group iv eyes, and 5 of 7 group v eyes were retained, and of the 32 eyes, 13 had visual acuity better than 20/200. eleven patients experienced a radiation - induced complication. no patient developed a second malignancy during follow - up. despite the limited number of patients enrolled , ebr may provide a mean of preserving eyeball and vision for some advanced lesions.

vascular complications include hematoma, pseudoaneurysm, arteriovenous (av) fistula, thromboembolism, arterial laceration and infection3,4,9 ). it is important to know the risk of complications and how to avoid possible causes that may cause such problems. the presence of an accessory renal artery is not uncommon variation, and this could function as the cause of unusual renal subcapsular hematoma. a 54-year - old female was admitted to our hospital for the evaluation of unruptured cerebral aneurysm. she had a 5-year history of diabetes mellitus and hypertension. eighteen months earlier, she had chest pain and underwent transfemoral cardiac angiogram at a different institute. since then, she had been taking aspirin 100 mg under the diagnosis of angina. she was diagnosed as having multiple aneurysms in both the anterior communicating artery and in the paraclinoid portion of the internal carotid artery. her blood pressure was 90/60 mm hg and the body temperature was 36.4. on physical examination, the abdomen was soft but tender on the left upper quadrant. laboratory findings were white blood cell 6100/mm, hb 10.8 g / dl, platelets 174000/mm. abdominal ct showed a left renal subcapsular hematoma in the lower pole portion (fig . six months later, her 64-cut multislice ct scan showed complete regression of the subcapsular hematoma ( fig . 2). interestingly, in her three - dimensional angiogram, small accessory renal arteries supplying the lower pole of the kidney were found at both sides (fig . diagnostic cardiac angiography with transfemoral catheterization has 1 - 2% risk of major complications5). hematomas are usually located in the femoral puncture site, retroperitoneal space, intraperitoneal region, and abdominal wall3,4 ). risk factors for cardiac catheterization hematoma formation are age older than 60 years, female, angioplasty instead of angiography alone, hypertension, thrombocytopenia, large - bore catheter size, operator inexperience, poor groin compression after sheath removal, high puncture site, peripheral vascular disease, and anticoagulant - thrombolytic therapy 3,4,9 ). neoplasm, bleeding tendency, vasculitis and infection are the causes of spontaneous bleeding. regarding iatrogenic causes, renal biopsy, extracorporeal lithotripsy, and anticoagulants are some sources. the incidence of iatrogenic renal artery injury during the percutaneous transluminal angioplasty is about 6.5 - 22.8%1,3 ). stable wire position and j - shaped guide - wire could avoid these vascular injury1 ). there has been only one paper in english describing renal subcapsular hematoma after femoral artery catheterization3 ). in that study , they reported that calcified plaques in the aorto - renal junction may get dislodged during catheter or wire passage. after dislodging of plaques, renal subcapsular hematoma could form through the dissected vessel3 ). typically, renal arteries branches at the level of the upper margin of the second lumbar vertebral body, 1 cm below the origin of the superior mesenteric artery6,7 ). one or two accessory renal arteries are found in about 23%, especially on the left side6,7,8 ). bilateral multiple renal arteries are found in 10% of the population7,10 ). in our case, 3 ). multidetector computed tomography is a very useful tool in most cases for the visualization of abdominal organs and vascular structures2,10 ). left accessory renal artery branches at the level of the lower margin of the third lumbar vertebral body and supplies the lower pole of the kidney. we used the introducing wire that was straight shape and 45 cm long (radifocus introducer ii, terumo co., tokyo, japan) for sheath placement. in small height person, relatively short distance from puncture site to opening of accessory renal artery could be possible. we used the 150-cm - long, 0.035-inch nitinol hydrophilic guide - wire with 45-degree angled, 3.0-cm soft tip (radifocus guide wire m, terumo co., tokyo, japan). the possibility of injury in the proximal portion of the left accessory renal artery during the wire insertion for sheath placement or the guide - wire passage around the level of the lower lumbar vertebral body was considered. the right femoral approach is the most commonly used access site for percutaneous angiography. in the case of the right side femoral approach, the left side accessory renal artery supplying the lower pole is prone to accidental damage. usually the right side accessory renal artery is located more distal than left and should be careful in case of left femoral puncture (fig . the previous report also involved a case of left lower pole subcapsular renal hematoma . furthermore , we were able to detect calcified plaques which might be located in the accessory renal artery supplying the lower pole of the kidney3). accessory renal arteries are not rare and easy to be injured at the level of the lower lumbar vertebral body. careful guide - wire passage under full visual inspection is important to avoid inadvertent vascular injury during percutaneous angiography.

vascular complications after percutaneous angiography include hematoma, pseudoaneurysm, arteriovenous fistula, thromboembolism, arterial laceration and infection. hematomas may occur in the groin, thigh, retroperitoneal, intraperitoneal, or abdominal wall. a 54-year - old female underwent percutaneous transfemoral angiography for the evaluation of cerebral aneurysm. renal subcapsular hematoma developed 3 hours after the procedure. renal subcapsular hematoma after percutaneous angiography is very rare. we investigated the possible causes of renal subcapsular hematoma. to avoid this rare complication, we need to perform guide - wire passage carefully from the beginning of the procedure under full visual monitoring.

world health organization (who) estimated that over 237 million people required treatment for schistosomiasis in 2010 with estimates of up to an additional 779 million at risk globally. schistosomiasis is endemic in 76 countries worldwide and besides malaria is the second important parasitic disease for public health. of the 662 million people infected worldwide, 85% are from africa. in tanzania, schistosomiasis is highly endemic, and its prevalence varies from one region to another, with a prevalence of up to 80% in highly endemic areas. intestinal schistosomiasis is caused by schistosoma mansoni, and infections are acquired by contact with freshwater containing parasite larvae. the disease is hyper - endemic in the great lake regions of east africa, owing to the favorable habitat for snails of the biomphalaria genus, which are the intermediate host. recent studies around lake victoria indicated that the prevalence in schoolchildren vary widely. found that 14% of 1-year - old children along the kenyan shores of lake victoria were s. mansoni positive, whereas odogwu et al. found a prevalence of 47.4% in children <3 years of age around lake victoria in uganda the prevalence in school - aged children in these areas could be as high as 86 - 90%. in children, schistosomiasis normally presents with generalized, non - specific signs and symptoms, making it difficult to identify disease - specific morbidity indicators and challenging to develop tools for assessing those indicators. over time , morbidity may progress from subtle manifestations such as anemia, to severe, debilitating, and irreversible conditions such as growth stunting, impaired cognitive development, increased susceptibility to co - infection, decreased quality of life, exercise intolerance, infertility, portal hypertension, and liver failure. who estimated that more than a billion people are chronically infected with soil - transmitted helminths (sths). ascaris lumbricoides, hookworms (ancylostoma duodenale and necator americanus), and trichuris trichiura are the most common sth species with global prevalence of about 1,000, 900 - 1,300, and 500 million people, respectively. as such, sth infections are still considered to be the most prevalent infections of mankind. nowadays , sth has been classified among the most prevalent neglected tropical diseases (ntds) as they persist exclusively in the poorest populations. findings from a study done among schoolchildren in a lake victoria shore line ward in tanzania showed that the prevalence of hookworms was the second to intestinal schistosomiasis. in kenya, the prevalence of sths is prominently attributed to a. lumbricoides, hookworms, and t. trichiura. hookworm infections observed in the lake basin in both tanzania and kenya were similarly high, ranging from 37.0 - 42.5%. other studies in magu district, tanzania, reported a prevalence of ascariasis of < 1%, and handzel et al. reported in kenya's nyanza province the prevalence of 22.9% and 17.9% for a. lumbricoides and possible factors associated with sth infections among the studied children included age (school - age), absence of toilet and piped water supply in the household, large family size (7 members), and not washing hands before eating and after defecation. although helminths can infect all members of a population, there are specific groups who are more vulnerable and at a greater risk of infection. for schistosomes and sths, the most vulnerable groups are preschool children, school - aged children, and women of child - bearing age, including adolescent girls although much of the morbidity associated with infection can be reversed with the use of effective anthelmintic drug treatments. preschool children and school - aged children including adolescents tend to harbor the greatest numbers of intestinal worms and schistosomes and as a experience growth stunting and diminished physical fitness as well as impaired memory and cognition. these adverse health consequences combine to impair childhood educational performance, reduce school attendance, with hookworms being well - known causes of anemia because of intestinal blood loss. chemotherapy with praziquantel (pzq) is currently the mainstay of control, which is available at a low cost. moreover, due to their geographical overlap between schistosomes and sth infections, they could be simultaneously treated with 2 drugs, albendazole and praziquantel. as such, the successful sth control programmes have enormous benefits of improved nutritional and health status of the children, higher educational attainment, labor force participation, productivity, and income among the most vulnerable populations. an added externality is the impact that ntds have on hiv / aids, tuberculosis, and malaria. several recent papers highlight the immunosuppressive features of helminths (especially the sths, schistosomes, and filariae) and their possible impact on promoting susceptibility to the big 3 diseases. new data suggest that control of ntds could become a powerful tool for combating hiv / aids, tuberculosis, and malaria. this paper presents data using a rapid assessment methodology to examine the prevalence of schistosomiasis and sth infections in schools around the tanzania perimeter of lake victoria. the study area is located on the northwest of tanzania around lake victoria (fig . 1). it stretches from the southwest of the lake through south to the eastern side. the area is comprised of kagera, mwanza, mara on the lake shore, and shinyanga about 60 km away from the lake. the study area is bordered by uganda and rwanda in the north and west, respectively. it is also bordered by tabora and manyara regions in the south and east, respectively. the main ethinic groups are; wahaya and wasubi in kagera region, wasukuma, wazinza, and wasumbwa in mwanza region, wajita, wakurya, and waluo in mara region, and again wasukuma and wasumbwa in shinyanga region. much of the activities have a bearing to schistosomiasis and sth infections as they performed without protective gear in the area where the level of sanitation and hygiene is as low as in any poor resource settings. collection of stool and urine specimens was done at the selected schools. each child was given 1 stool container and 1 urine container on the first day and asked to bring the urine and stool specimens. the smears were examined for hookworms and other helminth eggs or larvae within 1 hr after preparation in order to capture hookworm eggs before they hatch. the egg counts on each of the 2 slides were added together and divided by 2 to get the mean number of egg counts for the 2 slides. urine samples were collected after 10.00 a.m. to diagnose s. haematobium based on detection of eggs using the filtration technique. urine samples were mixed thoroughly and 10 ml were drawn using a syringe and passed through the membrane where eggs were logged. examination was done on site where detected eggs were counted and expressed as the number of eggs per 10 ml of urine. data entry was done using dbase iv (borland international, scotts valley, california, usa), and a double entry system was used for quality control. data were transferred to stata version 8 software (statacorp 2000, college station, texas, usa) for analysis. analysis was done by generation of some frequency tables, cross tabulations, and calculation of the prevalence. the ethical and scientific clearance was obtained from the medical research coordinating committee of the national institute for medical research before the implementation of the study. the study team visited villages and schools where community leaders and teachers were respectively met and the objectives, procedure, and potential harm and benefits of the study were explained to them. the leaders in turn convened meetings of the community members, and the same was explained to them before the consent to participate in the study was sought. any participant would be free to withdraw from the study at any time of the study period when he or she felt to do so. the decision to refuse or withdraw from the study would have no negative effect on the benefits provided during and after the study. all subjects who would be found infected with schistosomes and intestinal helminths were treated following standard treatment guidelines using praziquantel and albendazole tablets. participants were informed that information will be confidentially kept using code numbers instead of names of participants. the study area is located on the northwest of tanzania around lake victoria (fig . 1). it stretches from the southwest of the lake through south to the eastern side. the area is comprised of kagera, mwanza, mara on the lake shore, and shinyanga about 60 km away from the lake. the study area is bordered by uganda and rwanda in the north and west, respectively. it is also bordered by tabora and manyara regions in the south and east, respectively. the main ethinic groups are; wahaya and wasubi in kagera region, wasukuma, wazinza, and wasumbwa in mwanza region, wajita, wakurya, and waluo in mara region, and again wasukuma and wasumbwa in shinyanga region. much of the activities have a bearing to schistosomiasis and sth infections as they performed without protective gear in the area where the level of sanitation and hygiene is as low as in any poor resource settings. was given 1 stool container and 1 urine container on the first day and asked to bring the urine and stool specimens. the smears were examined for hookworms and other helminth eggs or larvae within 1 hr after preparation in order to capture hookworm eggs before they hatch. the egg counts on each of the 2 slides were added together and divided by 2 to get the mean number of egg counts for the 2 slides. urine samples were collected after 10.00 a.m. to diagnose s. haematobium based on detection of eggs using the filtration technique. urine samples were mixed thoroughly and 10 ml were drawn using a syringe and passed through the membrane where eggs were logged. examination was done on site where detected eggs were counted and expressed as the number of eggs per 10 ml of urine. data entry was done using dbase iv (borland international, scotts valley, california, usa), and a double entry system was used for quality control. data were transferred to stata version 8 software (statacorp 2000, college station, texas, usa) for analysis. analysis was done by generation of some frequency tables, cross tabulations, and calculation of the prevalence. the ethical and scientific clearance was obtained from the medical research coordinating committee of the national institute for medical research before the implementation of the study. the study team visited villages and schools where community leaders and teachers were respectively met and the objectives, procedure, and potential harm and benefits of the study were explained to them. the leaders in turn convened meetings of the community members, and the same was explained to them before the consent to participate in the study was sought. any participant would be free to withdraw from the study at any time of the study period when he or she felt to do so. the decision to refuse or withdraw from the study would have no negative effect on the benefits provided during and after the study. all subjects who would be found infected with schistosomes and intestinal helminths were treated following standard treatment guidelines using praziquantel and albendazole tablets. participants were informed that information will be confidentially kept using code numbers instead of names of participants. a total of 5,848 schoolchildren from 36 selected schools in the 4 regions of the lake victoria basin in tanzania were recruited for the study (table 1). the same numbers of girls and boys aged between 7 and 16 years from class 1 to 7 were selected for the study. intestinal schistosomiasis caused by s. mansoni was prevalent in 3 regions of kagera, mwanza, and mara. urogenital schistosomiasis caused by s. haematobium was found in children from 3 regions of mwanza, mara, and shinyanga but not in kagera. among the 3 regions, the highest prevalence (16.7%) the mean prevalence of both schistosomiasis and sths in all regions ranged from low to moderate ones (table 1). in kagera region , all the common intestinal helminths, namely s. mansoni, a. lumbricoides, t. trichiura, and hookworm infections were prevalent (table 2). bwina primary school in chato district recorded an extremely high prevalence of intestinal schistosomiasis of 86.5% followed by bunena in bukoba urban district with a prevalence of 16.8%, and other schools had a prevalence of less than 10%. the highest prevalence was 71.4% at runazi school followed by kiziramuyaga 59.5% and bwanga 37.9% (table 2). the prevalence of ascariasis was the highest at buzirayombo 33.3%, followed by bwanga 28.4%, bunena 25.6%, and kyenshama 25.6% (table 2). the prevalence of trichuriasis in the region was relatively low with a mean of 1.5% where kiziramuyaga had the highest prevalence of 7.1%. in mwanza region, 3 helminth species detected included s. mansoni, s. haematobium, and hookworms (table 3). the highest prevalence of s. mansoni infection was recorded at nyamikoma primary school that registered 79.2%, followed by bungonya 63.1% and nyakaliro 63.0% (table 3). bungonya primary school had the highest prevalence of hookworms of 36.9% followed by mwaginghi 34.0% and nyamikoma 21.3%. the highest prevalence (32.2%) of urogenital schistosomiasis was at bugogo primary school (32.3%), and mwaginghi and kigongo were the second with a prevalence of both 28.0%. lumeji was the fourth with 21.3% prevalence while the rest of schools had prevalence less than 10% (table 3). the distribution of schistosomiasis and sths in mara region was low relative to other regions (table 4). s. mansoni was highly prevalent at mwisenge 36.1%, nyamitwebili 30.2%, gamasara 15.9%, and minigo 11.7%. the prevalence was the highest (27.2%) at guta a school followed by minigo 11.7% and bulamba 2.7%. the prevalence of hookworms in mara region ranged from 2.4% at mwisenge to 19.3% at minigo primary school. the prevalence of trichuriasis was the lowest of all the helminths in region. in shinyanga region, the highest prevalence (30.3%) was noted at luhumbo school in shinyanga rural district seconded by 24.8% at songwa primary school in kishapu district. intestinal schistosomiasis was not noted in the whole study areas, whereas the hookworm prevalence was low; ranging from 0% at songwa school to 17.1% at bukomela school. no ascariasis nor trichuriasis cases were noted at any of the schools in shinyanga region. the showed that generally the prevalence of s. mansoni, s. haematobium, and sths are considerably high in the study areas.. however, the prevalence of s. mansoni in kagera region is very low with the exception of a few pockets in chato district which is currently in geita region. this could be attributed to the fact that lake shores in kagera region are open instead of bays when wind blows make the waves splash the shores making the place an unconduncive habitat for snail intermediate hosts. moreover, some of the shores in kagera region are formed by escarpments thus deep and less habitable by the snails. the situation is quite different from shores in chato district which is a bay thus most of its shores are not susceptible to strong winds. the distribution of s. haematobium and s. mansoni along lake victoria is largely related to the distribution of the intermediate hosts. along the shore of the lake, members of the genus biomphalaria, the intermediate host for s. mansoni, are common with populations living along the lake shores and islands being highly affected by s. mansoni as the risk of infection increases. apart from the fact that schistosomiasis is focal, the noted high prevalence of the disease in this district could be attributed to the presence of bays that experience less speedy winds and strong waves to the shores making the place unconduncive habitat for snails. the highest prevalence of s. mansoni at bwina primary school in kagera region could be attributed to the location of the study site. bwina ward is a thin peninsular with an average width of less than 2 km. as such, most of the area is surrounded by lake victoria water that is inhabited by biomphararia species. bwanga and kiziramuyaga localities are close to the lake area that has small bays that are conduncive areas for the snail hosts. the absence of urogenital schistosomiasis in kagera region could be due to the absence of ponds and streams, and the fact that no paddy cultivation is practiced in this area. the prevalence of sth infections in kagera region was relatively higher than that in other regions. this could be due to the difference in natural vegetations and gardens as a of rain variations. kagera is sunny and warm most of the year with cool evenings and rains occuring almost every morning from march through may. sths are highly affected by surface temperature, altitude, soil type, and rainfall. this has an implication of the region having the best breeding ground of sths as well as more people consuming semicooked vegetables that may carry helminth eggs. in mwanza region, the distribution of s. mansoni was similar to that of mara with more happenings close to the lake and s. haematobium on the hinterland. the high prevalence of intestinal and urogenital schistosomisiasis in the study area was a function of the distance from lake victoria, the former being more prevalent at localities close to the lake, whilist the latter is more so away from it. moreover, in areas where the prevalence of s. haematobium was high, there was a significant prevalence of hookworms. the findings are in line with other studies in magu district in mwanza region. the spatial distribution of s. haematobium is in accordance to the presence of ponds and streams as well as the wetness and warmth of the soil that are prerequisites for proliferation of the 2 helminths. we could not easily establish why there were no ascaris or trichuris infections in mwanza and shinyanga regions that are situated in the middle of the study area. difference in soil structure and texture as well as climate among the regions could be the reasons for the difference. kagera region on the west bordering southern uganda and mara region on the west which borders western kenya have similar prevalence of the sths as compared to mwanza and shinyanga regions. the prevalence of hookworm infections, 34% at mwaginghi in kwimba district, and 21.7% at nearby nyamikoma location in magu district, was similar to an ealier report in the same area. despite the fact that mara region is closer to the western province in kenya with similar types of sth infections this study recoded a mean regional prevalence ranging from 1% to 8% whereas studies in kenya s nyanza province reported the prevalence of 22.9% and 17.9% for a. lumbricoides and t. the high prevalence of intestinal schistosomiasis at mwisenge and nyamitwebili schools were associated with their being very close to the lake shore. the high prevalence of urogenital shistosomiasis at guta a and minigo could be ascribed to paddy fields, that are good habitats for the snail intermediate host of s. haematobium, that were enormous in the sourounding areas. in mara region, moreover, in areas where schistosomiasis was high, there was a significant prevalence of hookworms. this association was reported elsewhere; these findings are in line with that from magu district in mwanza region and in western kenya. as expected, the prevalence of intestinal schistosomiasis was extremely low in shinyanga region that is more than 150 km away from the lake but had the highest prevalence of urogenital schistosomiasis. the showed that schistosomisasis and 3 sths infections are co - endemic in lake victoria basin in tanzania with a high probability of polyparasitism in the study participants. the prevalence of s. mansoni, s. haematobium, and sths ranged from low to moderate in most parts of the study area. intestinal schistosomiasis was prevalent along the lake victoria shores and decreased with distance from it. conversely, the prevalence of urogenital schistosomiasis increased with distance from the lake. from the study findings this could be given in a participatory manner so that each member gets engaged in the learning process especially through peer educators. second, study teams need to conduct regular research in order to control incidence and reinfection rates in the area by carrying out regular treatment exercises. third, community - based provision of adequate safe water supply and sanitation facilities are imperative. water supply could be done through construction / drilling of wells at each sub - village, institutions, and health facility.

the objectives of this study was to conduct a survey on schistosomiasis and soil - transmitted helminth (sth) infections in order to come up with feasible control strategies in lake victoria basin, tanzania. depending on the size of the school, 150 - 200 schoolchildren were recruited for the study. duplicate kato - katz stool smears were prepared from each child and microscopically examined for schistosoma mansoni and sths. urine specimens were examined for schistosoma haematobium eggs using the filtration technique. after the survey, mass drug administration was done using praziquantel and albendazole for schistosomiasis and sths infections, respectively. a total of 5,952 schoolchildren from 36 schools were recruited for the study and had their stool and urine specimens examined. out of 5,952 schoolchildren, 898 (15.1%) were positive for s. mansoni, 754 (12.6%) for hookworms, 188 (3.2%) for ascaris lumblicoides, and 5 (0.008%) for trichuris trichiura. out of 5,826 schoolchildren who provided urine samples, 519 (8.9%) were positive for s. haematobium eggs. the revealed that intestinal schistosomiasis, urogenital schistosomiasis, and sth infections are highly prevalent throughought the lake basin. the high prevalence of intestinal and urogenital schistosomisiasis in the study area was a function of the distance from lake victoria, the former being more prevalent at localities close to the lake, whilst the latter is more so away from it. control of schistosomiasis and sths in the study area requires an integrated strategy that involves provision of health education to communities, regular treatments, and provision of adequate safe water supply and sanitation facilities.

linkage analyses performed in the early 1990s and centered on early onset alzheimer s disease families identified the only fully penetrant mutations known to date to be involved in this disease. three genes were found to carry these mutations: app, psen1 and psen2 (amyloid beta ( a4) precursor protein, presenilin 1, and presenilin 2 ). additionally, a strong risk factor was also identified through family studies: the e4 allele of apoe (apolipoprotein e) was found to increase the risk for alzheimer s disease in different populations. after these findings traditional approaches to the study of mendelian diseases relied on genetic linkage studies (a powerful tool to detect the chromosomal location of disease genes which is based on the observation that genes that reside physically close on a chromosome remain linked during meiosis). these studies involved large multi - generational pedigrees and needed the availability of multiple affected and unaffected individuals for testing.. when such families were available, genetic markers would be used to determine which areas of the genome only the affected individuals shared. this would provide evidence regarding the location of the causative gene, after which, dna sequencing, usually be means of the sanger method (based on the selective incorporation of chain - terminating dideoxynucleotides by dna polymerase during in vitro dna replication) would be performed to pinpoint the actual mutation. although a powerful approach, that has yielded many substantive findings, genetic linkage studies have a number of drawbacks that limit its utility: 1 ) large pedigrees are, in many cases, not available for study, particularly for late - onset diseases, where older generations have often died and descendants have not yet reached the age at onset; 2 ) the markers used to perform linkage, although dispersed throughout the genome, were usually in the hundreds, meaning that regions of linkage were very extensive and generally containing tens to hundreds of genes, having a clear impact on the costs of follow - up by sequencing; 3 ) the amount of time required to study a single family using this approach was quite significant, usually several months to years. more recently, the development of genome - wide genotyping and second - generation sequencing technologies has drastically changed the approaches used to study mendelian disorders. diseases presenting an autosomal recessive pattern of inheritance are well suited for autozygosity analysis (performed by high - density whole genome genotyping) and whole exome sequencing. in some cases, diseases presenting an autosomal dominant pattern of inheritance can now be studied by analyzing the exome variability in one or two affected family members without the need for linkage analyses. although linkage analyses will be valuable and in some cases essential, isolated exome sequencing analyses in small families with neurological diseases already ed in many significant findings. over 20 genetic loci with low risk effects for alzheimer s disease have been identified by gwas (table 1). these studies assess common genetic variability at the genome level between large groups of cases and controls. each of the loci identified contributes modestly to the risk of developing the disease, but new polygenic models are currently being investigated to try and establish more accurately who will develop alzheimer s disease. common variant hypothesis of human disease (for a description of gwas in human disease refer to) and the identification of the specific variant(s) and true effect(s) on the development of ad can only be achieved with fine mapping and functional analyses of each locus. it is also important to note that these loci seem to be part of a few biological pathways, indicating that these pathways, although it is currently not clear how, have an essential role in the pathobiological processes occurring in alzheimer s disease. three main pathways can be identified: the immune system and inflammatory responses; cholesterol and lipid metabolism; and endosomal vesicle recycling. for instance, neuropathological analyses of ad brains and functional assays connecting the amyloid beta protein to the complement pathway activation have given consistent evidence of the involvement of several complement cascade factors in the pathogenesis of this disease. another example is sorl1 (sortilin - related receptor with a - type repeats), which has been extensively studied in ad due to its role in app metabolism. sorl1 modulation of the app metabolism partially occurs in the endosomal pathway as part of a unique regulatory pathway for the control of neuronal protein transport. however, it has not been possible to undoubtedly associate variability in this gene to ad before the application of gwas to the study of late onset alzheimer s disease (load - alzheimer s disease occurring after 65 years of age). over 15 case - control association studies of sorl1 have produced positive, negative and trend associations between genetic variability at this gene and the risk of ad in different populations (alzgene assessed on april 2014 : http://goo.gl/xsrrl6). the initial gwas did not show a significant association between ad and the sorl1 locus. a significant association was only established in the recent meta - analysis using data from more than 70,000 people. the contradictory initial case - control reports may have been caused by different inclusion criteria used, with only cohorts tightly controlled for ethnicity achieving significance. also important to substantiate the association of this gene with ad was the identification of potentially pathogenic mutations in french early - onset ad (eoad alzheimer s disease occurring before 65 years of age) cases. in this study the authors performed exome sequencing in 14 autosomal dominant eoad index cases without mutations on the known ad genes and identified sorl1 mutations in 5 patients (1 nonsense mutation and 4 missense), which were not found in healthy controls. in a replication sample, including 15 autosomal dominant eoad cases, they identified 2 other novel mutations ing in a total of 7 mutations in 29 cases. although independent replications and segregation analyses are still needed, these indicate a potential pathogenic causative effect of sorl1 mutations in ad. the ability to sequence the whole genome, or more frequently, the whole exome of patients has led to a renewed interest in the study of mendelian forms of diseases and family studies. consequently, the potential contribution of rare genetic variants with strong effects in complex phenotypes like alzheimer s disease is currently under analysis. these technologies, when applied to a large enough number of cases and controls, also have the potential to identify moderate risk factors with lower allele frequencies in the population than the ones identified by gwas. variants with rare / moderate allele frequencies usually confer a greater risk for disease when compared with common variants due to purifying selection: high - risk alleles contributing to early - onset diseases may reduce reproductive fitness and consequently be driven to low frequencies. even though high risk alleles are rare in the population, these can be very informative by implicating genes with strong etiologic and biological roles in disease. these genes are usually the basis for functional genetic studies and considered important targets for the development of novel therapies. extended families are an extremely valuable asset for genetic studies since the same chromosomal segments recur in relatives. when studying late onset disorders it is frequently difficult to identify such families and by studying a large number of these families it seems more and more plausible that no other fully penetrant dominant genes like app and the psens genes are to be found in alzheimer s disease. some of these families will likely have private mutations as the cause of their pathological processes. by studying rare recessive diseases we have recently been able to uncover significant genetic factors involved in common disorders like alzheimer s and parkinson s diseases. by performing autozygosity and exome sequencing analyses in turkish dementia families we identified trem2 homozygous mutations causing frontotemporal dementia. trem2 had previously been associated with nasu - hakola disease, a rare autosomal recessive form of dementia presenting with bone cysts. inbred (and frequently isolated) populations are ideal for the study of disease genetics. inbreeding can increase the incidence of rare recessive disorders and isolated populations have an allelic load mostly derived from the alleles present in the population founders. rare recessive alleles with an effect in disease risk or causation tend to be located within long genomic regions of homo - zygosity. these long stretches of loss of heterozygosity can be easily assessed using snp arrays (fig . 1). this figure represents the from an illumina infinium whole - genome genotyping assay as visualized in genomestudio. it shows homozygosity mapping for three siblings (two affected by dementia and one unaffected). each blue dot represents one individual marker or single - nucleotide polymorphism (snp). for each snp , a low b allele frequency indicates that the individual is a homozygote for the a allele; intermediate values mean they are a heterozygote and high b allele frequency means that they are a homozygote for the b allele. this example shows a region of chromosome 4 (vertical blue area) in which the affected siblings present a large (over 7 mb) homozygous region that is not shared by the unaffected family member. this finding suggests the possibility that a disease - causing homozygous mutation may be present in this genomic region. this strategy combining autozygosity analysis with exome sequencing allowed us to identify trem2 mutations in cases presenting with an atypical phenotype to that previously associated with the gene. the 3 cases we identified by exome sequencing would have never had the trem2 gene sequenced by standard methods because of the atypical presentation. this clearly represents the power of whole - exome and whole genome sequencing in the establishment of molecular diagnoses. the identification of mutations in genes previously associated with different phenotypes has been a consistent finding from the application of exome sequencing to neurological disorders. these are critical to the understanding of the biological processes underlying these diseases by uncovering shared pathways. although load does not exhibit obvious recessive inheritance, recessive alleles may play a role as genetic risk components in this form of the disease. an excess of runs of homozygosity and larger homozygous tracts have been reported in load cases when compared to controls. in early - onset familial ad one mutation in app (p.a673v) has been reported to cause disease only in the homozygous state, whereas heterozygous carriers are unaffected, which is consistent with a recessive mendelian trait of inheritance. the heritability for load is estimated to be between 62 and 81 % and for eoad between 92 and 100 %. dominant causing mutations do not account for all this heritability and the belief that only highly penetrant dominant ad - causing mutations are in the basis of eoad is inconsistent with epidemiologic data that found apparent autosomal dominant transmission in approximately only 10 % of all eoad cases, leaving the majority of eoad unexplained. we have previously used whole - genome genotyping arrays to study two siblings from an israeli family consisting of seven siblings from a first - cousin marriage. at the time of the clinical study the mother was 90 years old with mild gait difficulties but otherwise healthy, and particularly presenting no cognitive loss. in this family we catalogued all the large regions of homozygosity shared between the two affected siblings and crossed this information with the genes known to have a possible role in ad. we are currently applying exome sequencing to follow up on these and pinpoint the mutation causing ad in this family. other ad families presenting with apparent autosomal recessive patterns of inheritance are starting to be described in the literature and we expect the study of recessive alleles in ad to have significant in the near future. following from the fact that our initial application of exome sequencing to the study of neurological diseases consistently identified mutations in genes previously associated with different phenotypes. and particularly after the identification of trem2 homozygous mutations causing frontotemporal dementia, we extended the genetic analyses of this gene to other forms of dementia. interestingly, when studying ad cases and controls we identified a heterozygous rare variant (p.r47h) that was significantly associated with an increased risk (or>3) for the development of ad. this was the first gene to be identified with a moderate risk effect on the disease since the association of the e4 allele of apoe was established for ad. taken together these clearly show that the study of recessive forms of rare neurological diseases can not only have a direct impact in the families where mutations are found, but can also have an impact on more common forms of diseases by contributing to the identification of risk alleles and, consequently, to a greater understanding of the pathobiological pathways underlying these disorders. trem2 is a transmembrane glycoprotein, consisting of an extracellular immunoglobulin - like domain, a transmembrane domain, and a cytoplasmic tail, which associates with dap12, also known as tyrobp. the association between these two proteins is required for its signaling function and it triggers the activation of immune responses in different immune cells. trem2 is known to control two streams of signaling to regulate the reactive microglial phenotype. one of these streams regulates phagocytosis and is associated with enhanced phagocytic pathways (which could be relevant to the removal of cell debris and the clearance of amyloid proteins in alzheimer s disease). this stream also regulates the alternative activation state of microglia, which is thought to be protective. the other signaling stream suppresses inflammatory reactivity and involves the repression of cytokine production and secretion. we believe that a reduced function of trem2 is key to the pathogenic effect of the risk variants associated with alzheimer s disease. trem2 is another gene known to be involved in inflammatory responses and the association found with alzheimer s disease corroborates the involvement of immunological pathways in this disease. this has led to an increased interest in the study of microglia (the immune cells in the brain) in these disorders. in ad, microglial cells are attracted to -amyloid plaques, produce elevated levels of proinflammatory cytokines and reactive oxygen species, and exhibit a change in morphology. interestingly, another ad risk gene (cd33) has recently been shown to inhibit microglial uptake and clearance of -amyloid. the analysis of the mendelian families with alzheimer s disease led to the identification of app and presenilin mutations and the formulation of the amyloid cascade hypothesis. until recently, this was the overarching hypothesis for nearly all attempts to develop therapeutics for the disease. these are ongoing but have had rather limited success as yet. the identification of the loci from gwas and of trem2 by sequencing has highlighted two other pathways, which may have therapeutic potential: the first is the innate immune system and microglia activation / inflammation and the second is brain cholesterol metabolism. this latter pathway has been long suspected because of the association of apoe with disease, but has become even clearer with the identification of abca7 as an alzheimer locus. both of these target pathways had been suspected before, but the identification of specific genes directly involved in pathogenesis will no doubt facilitate their closer examination. we are now in an unprecedented position to study genetic variability in mendelian and complex diseases due to the technologies developed in the last decade. the challenges ahead include the analyses of large datasets ing from studies of cases and controls but also the integration of these analyses with family studies. the assessment of alzheimer s disease families will most likely reveal a small number of new fully penetrant genes, but will be essential for the identification of novel moderate risk factors. collecting large cohorts of well - characterized samples will be essential to accurately dissect the molecular foundations of each locus associated with ad in order to understand its genuine contributions.

the analyses of genetic factors contributing to alzheimer s disease (ad) and other dementias have evolved at the same pace as genetic and genomic technologies are developed and improved. the identification of the first genes involved in ad arose from family - based studies, but risk factors have mainly been identified by studies comparing groups of patients with groups of controls. the best outcomes have been heavily associated with the capacity of interrogating genetic variability at the genome level without any particular a priori hypothesis. in this review we assess the role of genetic family studies in alzheimer s disease and other dementias within the current status of dementias and, particularly, ad s genetic architecture.

recent clinical studies have shown that treatment with angiotensin ii type 1 (at1) receptor blockers or angiotensin - converting enzyme (ace) inhibitors protects against the development of insulin resistance in hypertensive patients and new onset of diabetes in at - risk patients, indicating that the renin - angiotensin system (ras) especially tissue ras may contribute to the regulation of glucose metabolism. these functional local rass have been found in diverse organ systems such as the pancreas , heart , kidney, vasculature, adipose tissue , and skeletal muscle. the (pro)renin receptor (( p)rr ) cloned in 2002 was reported to be expressed in various tissues. its ligand, prorenin, is known to be activated without catalytic conversion into mature renin when combining with the (p)rr and alters the activation of extracellular signal - regulated kinase 1/2 (erk1/2). although aliskiren theoretically will block such prorenin - dependent ang generation, an alternative way to suppress this ang source is the infusion of the (p)rr blocker called the handle region peptide (hrp). its effectiveness in vivo is controversial, in part because of a wide variety of doses that has been applied, ranging from 0.1 mg / kg per 28 days to 1.0 mg / kg per day. moreover, the monosodium glutamate- (msg-) induced obese rat is a model associated with insulin resistance that may occur without the presence of type 2 diabetes, depending on the age at which the animals are studied. the administration of msg to newborn rats in distinctive lesions in hypothalamic arcuate nucleus (arc) neurons. the neuronal loss impairs insulin and leptin signaling and impacts energy balance as well as pituitary and adrenal activity. this study aimed to determine the effect of hrp with a large dose of 1 mg / kg / d on glucose status in the msg rats. all animal protocols were approved by the ethics committee of shantou university medical college. timed pregnant sprague - dawley rats neonatal male rats were injected subcutaneously with either 4 mg / g of msg (sigma aldrich, mo, usa) or nacl (1.87% solutions) as control. the control group of rats was given normal diet, whereas all of the msg rats were given high - energy diet (445.5 kcal/100 g, slaccas, shanghai, china). at age of 8 weeks, the msg - treated rats were divided into 4 groups including the msg - control group (msg group, n = 6), hrp treated group (msg - hrp group, n = 6), losartan treated group (msg - l group, n = 6), and losartan and hrp cotreated group (msg - hrp - l group, n = 6). then (day 0 and day 15) osmotic minipumps (2ml4 alzet, ca, usa) were implanted subcutaneously under isoflurane anesthesia to infuse vehicle (saline) or hrp (nh2-rillkkmpsv - cooh, chinapeptides, shanghai, china, 1 mg / kg per day, for the msg - hrp and msg - hrp - l group). the rats of msg - l group and msg - hrp - l group were given drinking water with 0.45 g / l losartan (merck, hangzhou, china). oral glucose tolerance test (ogtt) was performed after 16 h overnight fasting. glucose (2 g / kg) was administered orally, and a small amount of blood (about 100 ml) was collected from the tail vein at 0, 30, 60, and 120 min for insulin (elisa, cusabio, wuhan, china) and glucose measurement immediately with a glucometer (johnson & johnson, new brunswick, usa). for insulin tolerance test (itt), after 4 h of fasting, rats were given an intraperitoneal injection of 0.5 u / kg human insulin (novo nordisk, tianjin, china), and glucose was measured immediately with a glucometer at 0, 15, 30, and 90 min. sbp was measured in triplicate on separate occasions throughout the day, using the tail - cuff method (kent scientific copporartion, connecticut, usa) before the animals were sacrificed. at 12 weeks of age, body weight and length were measured, and lee's index was calculated according to the formula: lee's index = weight (g)31000/length (cm). after the performance of ogtt and itt, rats were sacrificed using pentobarbital sodium with the dose of 50 mg / kg weight. blood samples from puncturing heart were collected into edta tubes for the measurement of plasma ang - ii concentration. the pancreas was rapidly dissected out and bisected longitudinally, with one half snap frozen in liquid nitrogen and stored at 80c before use, and the other half fixed in 4% paraformaldehyde and embedded in paraffin. four - micron paraffin sections were prepared from 4% paraformaldehyde - fixed, paraffin - embedded rat pancreas. sections were stained with 0.1% sirius red (sigma aldrich, mo, usa) in saturated picric acid (picrosirius red) for 1 h and mounted. the ratio of stained area to the area of whole islet was calculated using the computer - imaging software ipp6.0. the expressions of the -cell marker insulin and -cell marker glucagon were examined by immunohistochemistry using insulin antibody (1 : 1,000 ; santa cruz, tx, usa) and glucagon antibody (1 : 100 ; santa cruz, tx, usa), respectively. after washing, a secondary antibody (1 : 500, biotin - conjugated goat anti - rabbit igg ; boster, wuhan, china) was applied for 30 min at room temperature. the ratio of stained area (-cell mass and -cell mass) to the area of whole islet was calculated using ipp6.0. the values were obtained from three islets in each section obtained from six rats in each group. proliferation of intraislet was assessed by immunohistochemical staining for proliferating cell nuclear antigen (pcna) using pcna antibody (1 : 100 ; santa cruz, tx, usa). specific immunohistochemical staining was detected using the streptavidin horseradish peroxidase and dab as the chromogen. semiquantitative assessment of intraislet proliferation was performed by determining the number of pcna - positive cells per islet section. pancreatic tissue was obtained and homogenized with tris - hcl buffer (ph 7.0). nadph oxidase activity was assayed using cytochrome c (genmed, shanghai, china). for assessing subunit of nadph oxidase p in islet sections, staining was performed for p (1 : 100 ; santa cruz, tx, usa). after washing, a secondary antibody (1 : 500, biotin - conjugated goat anti - rabbit igg ; boster, wuhan, china) was applied for 30 min at room temperature. the average gray - scale intensities of cells staining positively were measured by ipp 6.0. about 100 mg pancreas tissue was homogenized in 50 mmol / l tris buffer (ph 7.4), 150 mol / l nacl, 1% triton x-100, 1% sodium deoxycholate, 0.1% sds, and some inhibitors with a homogenizer on the ice and then centrifuged at 12000 rpm for 15 min at 4c. the concentration of ang - ii was measured by elisa (cusabio, wuhan, china) and the were corrected by the protein concentration. the msg - l group and the msg - hrp - l group had lower body weight than the msg group (p < 0.05). body length was decreased in the msg group compared with the con group and had no significant difference among msg rats received different treatment. lee's index and celiac adipose tissue wet weight, reflecting the extent of obesity, were increased in the msg group compared with the con group. the systolic blood pressure tended to increase in the msg group but had no statistical difference. the msg - l group and msg - hrp - l group had obviously lower systolic blood pressure than the msg group (p values were both < 0.01). serum ang - ii concentration had no significant difference between the con group and the msg group and was increased obviously in the msg - hrp - l compared with the msg group and the con group (p values were both < 0.01). the response of blood glucose to ogtt at week 12 of the experimental period was shown in figures 1(a) and 1(b). the blood glucose was higher in the msg - hrp group than the con group at 30 min after glucose load. the blood glucose in the msg - hrp group and the msg - hrp - l group were both higher than the con group at 60 min after glucose load (p values were < 0.01 and 0.05, resp .) and msg - hrp group was higher than the msg - l group (p < 0.05). the blood glucose in the con group was lower than the other four groups 2 h after glucose load and msg - l group lower than the msg - hrp group (p < 0.05). the area under the curve (auc) of blood glucose in the con group was lower than the msg group, msg - hrp group, and msg - hrp - l group (p values were < 0.05, < 0.01, and < 0.01, resp .) and had no statistical difference with the msg - l group, whereas the msg group was lower than the msg - hrp group (p < 0.05) and the msg - l group was lower than the msg - hrp group and the msg - hrp - l group (p values were both < 0.05). the response of serum insulin concentration to ogtt was shown in figures 1(c) and 1(d). the serum insulin concentration of the con group at fasting status, 30 minutes and 60 minutes after glucose load, and the auc of insulin were higher than the other four groups. the auc of insulin in the msg - l group was higher than the msg - hrp group (p < 0.05). insulin sensitivity was evaluated according to itt and the were shown in figure 2. the decreased rate of blood glucose was smaller in the msg group compared with the con group at 30 min after insulin injection (p < 0.01). treatment with hrp, losartan, and both hrp and losartan had higher decreased rate of blood glucose when compared with the msg group (p values were all < 0.05). islets cell and cell were marked by insulin antibody and glucagon antibody, respectively, according to immunohistochemistry. to quantify the change of -cell mass and -cell mass, the ratios of stained respective area of insulin and glucagon to the area of whole islet were calculated by ipp6.0 and the were shown in figure 3. the -cell mass in pancreas islets was reduced in msg rat when compared with con group (p < 0.05). treatment with hrp, losartan, and both increased -cell mass when compared with the msg group (p values were < 0.05, < 0.01, and < 0.01, resp .). for the -cell mass, treatment with losartan and both losartan and hrp reduced -cell mass when compared with msg group (p values were both < 0.01). cells staining positively for the pcna marker in pancreatic islets were shown in figure 4. the number of the pcna - positive staining cells was counted in the central area (70% of the central area of islet) and the peripheral area (30% area of the peripheral area of islets) of the islets according to ipp6.0. most pcna - positive staining cells were distributed in the periphery of the islets and were in accordance with the location of cell. compared with con group, the number was increased in the msg group (con versus msg : 12.15 7.23 and 95.00 9.04, p < 0.01). treatment with hrp had not changed the number of pcna - positive staining cells (90.25 12.37), whereas treatment with losartan decreased the number obviously (22.92 3.76, compared with msg group, p < 0.01). treatment with both hrp and losartan decreased the number obviously (43.35 14.25, compared with msg group, p < 0.01). there was no statistical difference for the pcna - positive staining cells in the central of the islets. the proteins of the pancreas tissue were extractted, and ang - ii was measured by elisa (figure 5). pancreas local ang - ii was obviously increased in the msg group compared with the con group (p < 0.01). the msg rats received treatment of hrp, losartan, and both hrp and losartan decreased the level of local ang - ii obviously compared with the msg group (p values were all < 0.01). fibrosis of the pancreatic islets was evaluated according to picrosirius staining (figure 6) and the ratio of stained area to the area of whole islets was calculated. the ratio was significantly increased in msg group (61.5% 8.92%) when compared with the con group (28.36% 6.84%). the msg rats received losartan (34.0 % 7.42%) and both losartan and hrp (35.6% 6.32%) treatment decreased the ratio obviously when compared with the msg group (p values were both < 0.01), whereas the msg rats received hrp treatment (53.0% 7.56%) had no statistical difference with the msg group. immunostaining of p in the pancreatic islets in five groups of animals was shown in figures 7(a)7(e) and the average gray - scale intensities were shown in figure 7(f). the immunostaining of p increased in the msg group when compared with the con group (p < 0.05). treatment with losartan, hrp, and both decreased the average gray - scale intensities of immunostaining of p (p values were < 0.05, < 0.01, and < 0.01, resp .). nadph oxidase activity in pancreatic tissue was increased in msg rats when compared with the control group. treatment with losatan, hrp, and both decreased the nadph oxidase activity in pancreatic tissue (compared with msg group, p values were all < 0.01). increased nadph oxidase activity was strongly correlated with levels of local ang - ii in pancreatic tissues (r = 0.665, p < 0.01). a large injection of sodium l - glutamate into newborn sd rats generated necrosis of neuronal cells of the ventromedial nucleus and arcuate nucleus in the hypothalamus, and as a , the rats developed polyphagia, obesity, and energy regulation barriers. our showed that msg rats given high - energy diet from 3 weeks age to 12 weeks led to obesity, insulin resistance, and elevated blood glucose level after glucose load. however, levels of insulin releasing after glucose load just reached about half of the normal sd rats. the above demonstrated that msg rat was a model with insulin resistance and -cell impairment. nemeroff et al. showed that msg rats had smaller endocrinic organs such as pituitary, testis, ovarian, adrenal, and lower levels of thyroid hormone and gave us a cue that -cell impairment may be a of dysplasias of pancreas islets. although serum insulin concentration was obviously lower than the normal sd rats, the msg rats had elevated blood glucose after glucose load but kept normal fasting blood glucose at age of 12 weeks and may due to relative low levels of antergic hormone of insulin such as glucocorticoids and thyroid hormone. the characteristic of glucose status of the msg rats supplied us with an ideal model for the study. showed that the rat pancreas expressed the major ras component genes, notably angiotensinogen and renin, required for intracellular formation of angiotensin ii. and lam and leung testified the presence of the at1 receptor, at2 receptor, angiotensinogen and (pro)renin in the human cell of the islets. our showed that (pro)renin, angiotensinogen, and (p)rr were detected in pancreatic islets, and the level of local ang - ii was independent of system ang - ii. the components of local ras are responsive to various physiological and pathophysiological stimuli such as hyperglycemia and lead to aggravation of islets functions in turn. activation of pancreatic local ras increased in different type 2 diabetes animal models including db / db mice, zdf rats, and oletf rats. our showed that level of pancreatic local ang - ii was remarkedly increased in msg rats. pretreatment of isolated db / db islets with losartan before the addition of angiotensin ii (100 nmol / l) not only completely rescued glucose - induced insulin secretion but also tended to increase insulin release to an even higher level. treatment with perindopril or irbesartan treatments significantly improved first - phase insulin secretion in zdf animals. liskiren decreased body weight and plasma glucose level and increased plasma insulin level in a fed condition. our showed that losartan increased the levels of insulin releasing after glucose load and decreased auc of blood glucose. much to our surprise, hrp improved insulin sensitivity but had not increased insulin releasing and had not improved glucose status. the maintenance of the specialized architecture of the pancreatic islet and normal -cell mass is important for continuing function. candesartan increased -cell mass and increased staining intensity of insulin in pancreas islets of db / db mice. treatment with aliskiren restored the -cell mass to a similar level to that in nondiabetic normal (c57bl/6j) mice. our indicated that losartan increased -cell mass and decreased -cell mass in accordance with the of insulin releasing test. the regulation of islet cell apoptosis and proliferation is important in maintaining normal ratio of -cell mass and -cell mass. our showed pcna - positive staining cells distributed in the periphery of the islets in accordance with cells in msg rats. losartan but not hrp decreased the pcna - positive staining cell and may be the reason why hrp had not improved glucose status of the msg rats. pancreatic islets are highly susceptible to oxidative injury, owing to low endogenous antioxidant activity. blockade of the ras with perindopril or irbesartan significantly reduced staining for nitrotyrosine in zdf rats. candesartan decreased staining intensity of components of nad(p)h oxidase, p, gp, and those of oxidative stress markers in -cell of db / db mice. treatment with losartan, hrp, and both decreased the average gray - scale intensities of immunostaining of p and nadph oxidase activity in pancreatic tissue. the above supported the notion that ras inhibitors improve islets functions by decreasing activity of oxidative stress. both perindopril and irbesartan reduced expression for collagen i and iv protein in zdf rats. islet fibrosis and the expression of tgf- with its downstream signal molecules were significantly reduced in the pancreas of oletf ramipril - treated group than in control group. demonstrated that hrp decreased the expression of collagen i and iii in the heart and collagen iv in the kidney in spontaneously hypertensive rats, hrp had not improved status of islets fibrosis in msg rats. from the above we can conclude that hrp and losartan had some similar effects on islets in msg rats. losartan decreased level of local ang - ii, increased -cell mass, and decreased the activity of oxidative stress. hrp had no effect on the -cell mass and proliferation of islet cells and had not improved status of islets fibrosis in msg rats. these difference may be due to the specially interaction of renin, prorenin, and (p)rr. renin is an aspartyl protease that cleaves angiotensinogen into angiotensin i, the rate - limiting reaction in the cascade generating angiotensin. both renin and its inactive precursor, prorenin, can bind to the (p)rr. the (p)rr is a true receptor that is able to activate intracellular signaling, and (p)rr bound prorenin is enzymatically active as a of a conformational change without cleavage of the prosegment. as a blocked of (p)rr, hrp decreased local level of ang - ii, and in the end, oxidative stress was decreased as it is ang - ii dependant. however, whether the activation of erk1/2 mapk pathway which was related to proliferation and fibrosis was blocked by hrp needs further investigation. in summary, on the one hand, both losartan and hrp decreased levels of pancreatic local ang - ii and nadph oxidase activity as well as its subunits p. on the other hand, losartan but not hrp decreased -cell mass and number of pcna - positive cells located periphery of the islets and decreased picrosirius red stained area in islets. hrp ameliorating insulin resistance but not -cell functions leads to hyperglycemia in the end in male msg rats, and the dual characters of hrp may partly account for the phenomenon.

handle region peptide (hrp), which was recognized as a blocker of (pro)renin receptor (( p)rr ), may block the function of (p)rr. the aim of this study was to investigate the effect of hrp with a large dose of 1 mg / kg / d on glucose status in the rats treated neonatally with monosodium l - glutamate (msg). at the age of 8 weeks , the msg rats were randomly divided into msg control group, hrp treated group with minipump (msg - hrp group), losartan treated group (msg - l group), and hrp and losartan cotreated group (msg - hrp - l group) and fed with high - fat diet for 4 weeks. losartan but not hrp increased the levels of insulin releasing and ameliorate glucose status although both losartan and hrp improved insulin sensitivity. on the one hand, both losartan and hrp decreased levels of pancreatic local ang - ii and nadph oxidase activity as well as its subunits p22phox. on the other hand, losartan but not hrp decreased -cell mass and number of pcna - positive cells located periphery of the islets and decreased picrosirius red stained area in islets. hrp ameliorating insulin resistance but not -cell functions leads to hyperglycemia in the end in male msg rats, and the dual characters of hrp may partly account for the phenomenon.

a modified gyrb cdna fragment was generated as previously described and ligated at the 3 end of a human l - selectin cdna via an xbai site introduced at the l - selectin translation - termination codon. all constructs were verified by dna sequencing, subcloned into the pmt-2 expression vector (provided by genetics institute, cambridge, ma), and used to transfect 300.19 cells. transfected cells were selected in rpmi 1640 medium containing 10% calf serum and g418 (1 mg / ml ; sigma chemical co., st . multiple clones of transfected cells expressing similar cell - surface levels of wild - type l - selectin or l - selectin gyrb fusion proteins were identified by immunofluorescence staining with flow cytometry analysis . cells were washed once with rpmi 1640 medium before incubation at 37c for 25 min ( unless indicated otherwise) in rpmi 1640 containing either 0.1% dmso or the indicated amounts of coumermycin and novobiocin (in 0.1% dmso ; sigma chemical co.). after washing with ice - cold pbs without ca / mg, the cells were divided and incubated with biotin - labeled ppme (5 g / ml) in pbs containing either ca/ mg or 10 mm edta. after a 30-min incubation on ice, fitc - labeled avidin was added to visualize phosphomanan monoester core complex (ppme) binding as previously described (3, 4), with staining assessed immediately by flow cytometry as in fig. ca - dependent ppme - binding was calculated by subtracting the mean linear fluorescence channel number for staining (in 10 mm edta) from the mean value of fluorescence staining in the presence of ca. antibiotic treatment did not change the mean fluorescence intensity of staining. for ligation with antibody, cells (2 10 cells / ml in flow medium, pbs containing ca / mg and 0.5% bsa) were incubated with lam1 - 101 or lam1 - 118 mabs (10 g / ml) at room temperature for 15 min. in vitro rolling experiments were as previously described using a transformed human umbilical vein endothelial cell (huvec) line (ea.hy926, provided by dr . cora - jean edgell, university of north carolina at chapel hill ; reference 14) that was transfected with an 1,3fucosyltransferase - vii cdna (fucosyltransferase vii, provided by dr . brent weston, university of north carolina at chapel hill). transfected ea.hy926 cells were grown to confluence on 25-mm circular glass coverslips and mounted in a parallel - plate flow chamber. flow medium was drawn through the chamber at a rate of 804 l / min with a syringe pump (harvard apparatus, natick, ma), which generates an estimated wall shear stress of 1.85 dynes / cm. cells (10 cells / ml) were perfused through the chamber for a 10-min period. cell rolling was observed using an inverted phase - contrast microscope (olympus corporation, lake success, ny) and videotaped using a ccd video camera (hitachi denshi, ltd ., tokyo, japan) with a supervhs video recorder (model svo-9500md ; sony corporation of america, new york, ny) and an attached time - date generator (microimage video sales co., bechtelsville, pa). interacting cells (tethering and rolling) were determined by analysis of videotapes in which four fields (0.16 mm) on a video monitor were counted at 14 random time points throughout the flow period. for calculating velocities, the distance each cell traveled between two time points was measured, converted into actual distance, and divided by the elapsed time. a modified gyrb cdna fragment was generated as previously described and ligated at the 3 end of a human l - selectin cdna via an xbai site introduced at the l - selectin translation - termination codon. all constructs were verified by dna sequencing, subcloned into the pmt-2 expression vector (provided by genetics institute, cambridge, ma), and used to transfect 300.19 cells. transfected cells were selected in rpmi 1640 medium containing 10% calf serum and g418 (1 mg / ml ; sigma chemical co., st . multiple clones of transfected cells expressing similar cell - surface levels of wild - type l - selectin or l - selectin gyrb fusion proteins were identified by immunofluorescence staining with flow cytometry analysis . cells were washed once with rpmi 1640 medium before incubation at 37c for 25 min ( unless indicated otherwise) in rpmi 1640 containing either 0.1% dmso or the indicated amounts of coumermycin and novobiocin (in 0.1% dmso ; sigma chemical co.). after washing with ice - cold pbs without ca / mg, the cells were divided and incubated with biotin - labeled ppme (5 g / ml) in pbs containing either ca/ mg or 10 mm edta. after a 30-min incubation on ice , fitc - labeled avidin was added to visualize phosphomanan monoester core complex (ppme) binding as previously described (3, 4), with staining assessed immediately by flow cytometry as in fig. ca - dependent ppme - binding was calculated by subtracting the mean linear fluorescence channel number for staining (in 10 mm edta) from the mean value of fluorescence staining in the presence of ca. cells were treated with dmso, coumermycin, and/or novobiocin as described above. for ligation with antibody, cells (2 10 cells / ml in flow medium, pbs containing ca / mg and 0.5% bsa) were incubated with lam1 - 101 or lam1 - 118 mabs (10 g / ml) at room temperature for 15 min. in vitro rolling experiments were as previously described using a transformed human umbilical vein endothelial cell (huvec) line (ea.hy926, provided by dr . cora - jean edgell, university of north carolina at chapel hill ; reference 14) that was transfected with an 1,3fucosyltransferase - vii cdna (fucosyltransferase vii, provided by dr . brent weston, university of north carolina at chapel hill). transfected ea.hy926 cells were grown to confluence on 25-mm circular glass coverslips and mounted in a parallel - plate flow chamber. flow medium was drawn through the chamber at a rate of 804 l / min with a syringe pump (harvard apparatus, natick, ma), which generates an estimated wall shear stress of 1.85 dynes / cm. cells (10 cells / ml) were perfused through the chamber for a 10-min period. cell rolling was observed using an inverted phase - contrast microscope (olympus corporation, lake success, ny) and videotaped using a ccd video camera (hitachi denshi, ltd ., tokyo, japan) with a supervhs video recorder (model svo-9500md ; sony corporation of america, new york, ny) and an attached time - date generator (microimage video sales co., bechtelsville, pa). interacting cells (tethering and rolling) were determined by analysis of videotapes in which four fields (0.16 mm) on a video monitor were counted at 14 random time points throughout the flow period. for calculating velocities, the distance each cell traveled between two time points was measured, converted into actual distance, and divided by the elapsed time. whether the cytoplasmic domain of l - selectin enhances ligand binding through oligomerization was tested directly by assessing the functional activity of induced cell - surface l - selectin dimers. although not previously tested for inducing dimerization of transmembrane proteins, the exogenous dimeric antibiotic coumermycin can cross - link and activate cytoplasmic raf-1gyrb fusion proteins by simultaneously binding two gyrb subunits. cdnas encoding l - selectin and a gyrb subunit were fused (l - gb) and expressed in 300.19 cells (fig . 1), an l - selectin negative leukemia cell line that positions l - selectin at microvillus tips when expressed. the interaction of l - selectin with its ligand was assessed using a multivalent mannose-6 phosphate - rich polysaccharide mimetic, ppme, which can be labeled to assess l - selectin binding activity without the influence of other adhesion receptors. cells expressing l - gb or wild - type l - selectin bound ppme similarly (fig . 2 a, top). coumermycin treatment significantly increased the ppme binding activity of l - gb expressing cells by three- to five - fold but had no effect on wild - type l - selectin bearing cells (fig . edta inhibited ppme binding by all cells, consistent with the involvement of l - selectin 's calcium - dependent lectin domain . treatment of cells with the l - selectin function - blocking mab, lam1 - 3, also blocked ppme binding ( data not shown). coumermycin enhancement of ppme binding by l - gb cells was dose dependent, maximal at 0.9 m coumermycin, rapid, and sustained (fig . pretreatment of l - gb cells with novobiocin, the monomeric coumermycin analogue, completely eliminated the coumermycin - induced effect (fig . coumermycin - induced dimerization did not increase l - gb expression levels or induce l - selectin endoproteolytic release from the cell surface ( fig . 2 e), whereas phorbol esters induced l - gb and wild - type l - selectin endoproteolytic release similarly (data not shown). coumermycin - induced l - selectin dimerization in l - gb cells did not generate transmembrane signals leading to upregulated intercellular adhesion over a 24-h time period. although the molecular explanation for why cross - linking l - selectin with some mabs induces potent homotypic adhesion while coumermycin does not induce homotypic adhesion is not known, cross - linking l - gb or wild - type l - selectin on expressing cells with appropriate anti l - selectin mabs induced potent homotypic adhesion (data not shown) as previously described. therefore, coumermycin - induced dimerization of l - gb ed in enhanced l - selectin binding activity specific for its ligand mimetic, ppme, which provides a mechanistic explanation for leukocyte activation rapidly upregulating l - selectin functional activity. the physiological importance of l - selectin dimerization for leukocyte / endothelial interactions was assessed using in vitro flow chambers that mimic vascular flow conditions in vivo. l - selectin supports leukocyte rolling on a monolayer of transformed huvec that express l - selectin ligand(s) . both l - gb and wild - type l - selectin mediated a basal level of 300.19 cell rolling (fig . 3, a and c), but neither cell type arrested on these huvec monolayers due to the absence of other operable adhesion molecules expressed by this cell line (data not shown). mediated rolling of cells on huvec monolayers was significantly enhanced (by > 700%, p < 0.002, n = 3 experiments) by coumermycin treatment, which was competitively inhibited by novobiocin pretreatment (fig . all rolling on huvec monolayers was completely blocked by the lam1 - 3 mab ( fig . 3, a and c). remarkably, coumermycin treatment also significantly lowered rolling cell velocities by 35%, from a median (sem) rolling velocity of 272 9 to 175 12 m / s (p < 0.001 ; n = 3, fig therefore, l - selectin dimerization markedly increased the number of leukocytes rolling under conditions of physiologic shear stress . to further verify that l - selectin dimerization enhances its functional activity, a panel of igg mabs was screened to identify those that cross - linked l - selectin extracellular domains in a functionally appropriate configuration while not blocking ligand binding or inducing transmembrane signals . of 32 mabs screened, the lam1 - 118 mab reactive with the short consensus repeat domains of l - selectin fit these criteria. lam1 - 118 mab treatment of wild - type l - selectin or l - gb bearing cells significantly increased the frequency (310 and 350% increase, respectively ; p < 0.01) of cells interacting with huvec monolayers under physiologic flow conditions (fig . 3 c). this was consistent with an increase in high endothelial venule binding by lymphocytes pretreated with this mab. as with coumermycin treatment of l - gb cells, lam1 - 118 mab treatment significantly lowered rolling cell velocities by 35%, from a median (sem) of 266 3 to 172 6 m / s (p < 0.001, n = 3 ; fig . lam1 - 118 mab treatment of wild - type l - selectin bearing cells lowered rolling cell velocities similarly ( data not shown). lam1 - 118 mab treatment of l - gb or wild - type l - selectin transfected cells did not increase or decrease receptor expression levels (data not shown), and did not induce homotypic adhesion of 300.19 cells. furthermore, treatment of wild - type or l - gb l - selectin bearing cells with lam1 - 101, an isotype - matched mab that binds the epidermal growth factor like / short consensus repeat domains of l - selectin, had no significant effect on cell attachment or rolling velocities (fig . neither 300.19 cells alone nor wild - type l - selectin expressing cells treated with the lam1 - 3 mab showed any detectable interaction with the huvec monolayers ( fig therefore, coumermycin - mediated cross - linking of l - selectin cytoplasmic domains and mab - mediated cross - linking of the l - selectin extracellular domains both generated identical under physiological flow conditions . l - selectin binds its ligands with rapid association and dissociation rates, which in a minimal shear stress threshold requirement for the initiation and maintenance of leukocyte rolling. despite the coumermycin- induced increase in l - selectin binding activity, both coumermycin - treated and -untreated l - gb cells required shear stress for the promotion of l - selectin dependent interactions since no tethering or rolling was observed below wall shear stresses of 0.75 dynes / cm (fig . 4). nonetheless, coumermycin - treated l - gb cells interacted with huvec monolayers at a significantly greater frequency compared with untreated l - gb cells at shear stresses between 0.75 and 3.0 dynes / cm (fig . thus, l - selectin dimerization influences rolling velocity and receptor detachment rates as opposed to selectin localization to the tips of microvilli, which appears to promote cell capture. l - selectin dimerization may retard the dissociation of selectin bonds, which would enhance the lifetime of l - selectin binding to its endothelial ligand(s) and promote leukocyte tethering to endothelium during rolling. that inducing l - selectin dimerization by either coumermycin or mabs can enhance l - selectin adhesion is consistent with a model in which the transient activation- enhanced adhesive function of l - selectin from receptor dimerization. however, these do not exclude other mechanisms for transiently enhancing the adhesive function of l - selectin and do not necessarily prove that l - selectin dimerization is a physiologic process. nonetheless, the formation of cell - surface dimers may also provide a mechanistic explanation of why l - selectin mediated tethers operate at high shear forces. thus, the rapid upregulation of l - selectin binding activity after leukocyte activation and l - selectin dimerization may stabilize l - selectin bonds under shear force, which facilitates the formation of a second receptor / ligand bond before the first one breaks during rolling. multivalent l - selectin binding would also distribute the tensile force applied on each tether among several l - selectin / ligand bonds. therefore, l - selectin's cytoplasmic domain and cytoskeletal associations may be required for its oligomerization within the cell membrane, providing it with strong resistance to shear stresses. whether dimerization also regulates p- and e - selectin function is unknown, although p - selectin isolated from activated platelets is found in a tetrameric configuration, which facilitates its binding activity in vitro. since leukocyte rolling may involve multivalent binding , rapid oligomerization of l - selectin would favor the formation of multivalent bonds with its low affinity endothelial cell ligands. consistent with this notion, l - selectin ligands consist of multimeric sialylated and sulfated oligosaccharides appropriately presented by mucin scaffolds . as such, oligomerized l - selectin molecules may interact cooperatively with ligands presenting multiple low affinity oligosaccharide binding sites that are optimally stabilized by multivalent bonding. this is consistent with the many animal lectins that dramatically increase their affinity for carbohydrate ligands by combining multiple oligosaccharide binding sites in each lectin polypeptide. however, in the case of l - selectin the generation of multimeric binding by receptor oligomerization may provide a rapid means for upregulating adhesion receptor function with leukocyte activation. in addition, dimerization may be particularly important when l - selectin or its ligands are expressed at low site densities. therefore, this study supports the notion that selectin oligomerization is of primary physiologic significance and is likely to directly influence leukocyte migration and entry into sites of inflammation. moreover, the coumermycin gyrb dimerization strategy is likely to be useful for studying other transmembrane proteins and adhesion molecules that share the property of being functionally upregulated in response to cellular activation. generation of l - selectin expressing cell lines. (a) structure of l - selectin and the l - selectin gyrb (l - gb) fusion protein containing the entire l - selectin protein in - frame with the nh2-terminal 24-kd subdomain of the b subunit of bacterial dna gyrase (gyrb). domains: egf, epidermal growth factor like; scr, short consensus repeat; tm, transmembrane; cyto, cytoplasmic. (b) cell - surface expression of wild - type l - selectin or l - gb in stably - transfected 300.19 cells and wild - type l - selectin expression by human blood lymphocytes. cells were isolated and stained with fitc - conjugated lam1 - 116 mab specific for l - selectin (solid line) or an isotype - matched, nonbinding control mab (dashed line) as previously described. fluorescence histograms from flow cytometry analysis are on a three - decade log scale and are representative of from at least five experiments. coumermycin - induced changes in ppme binding activity by l - gb expressing 300.19 cells. (a) immunofluorescence analysis of ppme binding by cells expressing wild - type l - selectin or l - gb, before and after treatment with 0.9 m coumermycin. after coumermycin treatment, the cells were incubated with ppme in the presence of either ca or edta, and ppme binding was assessed by fluorescence staining and flow cytometry analysis with shown on a three - decade log scale. these represent those obtained in at least five experiments with the l - gb clone shown (fig . 1 b) and are representative of obtained with two independent clones of l - gb transfected cells. (b) dose response of coumermycin - induced ppme binding in l - gb cells. values represent the mean fold increase in ppme binding relative to untreated cells obtained in four experiments. asterisk indicates significant differences between treated and untreated samples, p < 0.01, student's t test. (c) time kinetics of coumermycin - induced ppme binding by l - gb transfectants. the cells were treated with 0.9 m coumermycin for the indicated amounts of time before ppme staining. cells were first treated with medium or the indicated amounts of novobiocin at 37c for 15 min. coumermycin (0.9 m final) was then added with ppme binding assessed 25 min later. l - gb cells were incubated in media containing dmso (0.1%), 0.9 or 9.0 m coumermycin at 37c for the indicated time periods. (a) effect of coumermycin and novobiocin treatments on the number of l - gb cells rolling on a huvec monolayer in an in vitro flow chamber assay. values represent the number of l - gb cells interacting with huvec monolayers in a 0.16-mm field. (b) effect of coumermycin (0.9 m) on rolling velocities of l - gb cells interacting with a huvec monolayer. each symbol represents the velocity of an individual cell plotted in rank order with median (50%) velocities indicated by horizontal and vertical lines. (c) effect of anti l - selectin mabs on the number of wild - type or l - gb cells rolling on huvec monolayers. l - selectin mabs on the rolling velocities of l - gb cells interacting with huvec monolayers. a and c values are mean sem of obtained in three experiments, and b and d values are representative of obtained in three experiments. l - selectin dimerization enhances leukocyte attachment and rolling on endothelial cells under physiologic flow. wall shear stress was varied at 1-min intervals by changing the flow rate through the flow chamber. asterisk indicates coumermycin - treated cells that were significantly different from untreated cells, p < 0.05. values represent means sem of from three experiments.

l - selectin binding activity for its ligand expressed by vascular endothelium is rapidly and transiently increased after leukocyte activation. to identify mechanisms for upregulation and assess how this influences leukocyte / endothelial cell interactions, cell - surface dimers of l - selectin were induced using the coumermycin gyrb dimerization strategy for cross - linking l - selectin cytoplasmic domains in l - selectin cdna - transfected lymphoblastoid cells. coumermycin- induced l - selectin dimerization ed in an approximately fourfold increase in binding of phosphomanan monoester core complex (ppme), a natural mimic of an l - selectin ligand, comparable to that observed after leukocyte activation. moreover, l - selectin dimerization significantly increased (by 700%) the number of lymphocytes rolling on vascular endothelium under a broad range of physiological shear stresses, and significantly slowed their rolling velocities. therefore, l - selectin dimerization may explain the rapid increase in ligand binding activity that occurs after leukocyte activation and may directly influence leukocyte migration to peripheral lymphoid tissues or to sites of inflammation. inducible oligomerization may also be a common mechanism for rapidly upregulating the adhesive or ligand - binding function of other cell - surface receptors.

the ev surveillance system in south korea consists of 62 clinics (8 primary clinics, 14 secondary hospitals, and 40 tertiary hospitals located nationwide) managed by pediatric physicians (figure). during 2009, a total of 2,427 cases of viral disease were reported to the korea centers for disease control and prevention through a web - based system. in addition, an experienced neurologist (w .- s.r .) collected detailed clinical information about, and of imaging studies of, patients reported to have central nervous system (cns) involvement. we monitored the patients until they were discharged or for 3 weeks if duration of hospitalization was > 3 weeks. patient outcome was classified into 1 of 4 groups; no sequelae (neurologic dysfunction without dependency), mild sequelae, severe sequelae (neurologic dysfunction requiring assistance), or death. geographic distribution of clinics participating in enterovirus surveillance, south korea, 2009. pcr (rt - pcr) by using taqman technology (applied biosystems, foster city, ca, usa). briefly, viral rnas were extracted by using the magnetic bead based viral nucleic acid purification protocol described by boom et al. subsequently, 1-step real - time rt - pcr was performed by using a dual - labeled fluorogenic ev - specific probe and primers designed on the basis of previous data. for genotyping, seminested rt - pcr was used to amplify part of the viral protein (vp) 1 gene of ev, based on the korea centers for disease control and prevention protocol for detection of pan - ev, and sequencing analysis for vp1 amplicon was performed by using automatic sequencer and dnastar software package. in 2009, ev was detected in 461 (19%) of all patients and in 321 (66%) of patients with suspected hfmd and herpangina. samples from 331 (72%) of the 461 ev - seropositive patients were available for genotyping (table 1). in addition, we found 112 cases of hfmd with cns complications (meningitis or encephalitis); ev was detected in 95 (85%) and ev71 in 92 (82%) case - patients. furthermore, ev71 was detected in 2 of the 187 case - patients in which meningitis without hfmd or herpangina was diagnosed. mean (sd) patient age was 46 months (range 1 month12 years); 12 (13%) patients were < 1 year of age. initial diagnoses were viral meningitis (60 patients), encephalitis, acute cerebellar ataxia, acute transverse myelitis, and guillain - barr syndrome. rash and fever median leukocyte count was 111 cells / mm (interquartile range 48318 cells / mm), mean (sd) protein level was 43 mg / dl, and mean (sd) glucose level was 69 mg / dl. of the 32 (34%) patients who underwent magnetic resonance imaging of the brain, 24 (75%) had normal , 4 (13%) had meningeal enhancement on t1-enhanced imaging, and 4 (13%) had high signal intensity at the brainstem or cerebellum on t2-weighted or fluid - attenuated inversion recovery imaging. all brain parenchymal lesions were located in the brainstem or cerebellum, and the 4 patients with brain lesions subsequently experienced ataxia. ev71 was found in 72 (92%) of 78 lower gastrointestinal tract samples, 37 (60%) of 62 upper respiratory tract samples, and 2 (5%) of 37 csf samples. on the basis of sequence analysis, c4a, with high similarity to strains from china in 2008, was a dominant serotype of ev71 (76%); and c1 was found in 2 patients and c5 in 1 patient. the partial vp1 sequences of korean ev71 strains were registered in genbank (hm443164644), and viral genetic identity belonged to c4a genotype, which was not a prevailing genotype in the previous reports of other asian pacific countries. * hfmd, hand - foot - and - mouth disease; ev71, enterovirus 71; prc, people s republic of china; na, not available. the severity of, and case - fatality rate for, ev71 infection in our population were relatively low compared with those of previous reports in which the case - fatality rate ranged from 10% to 26%. in contrast, the predominant genotype in our patients was c4, particularly c4a, which has been prevalent in china since 2008. the case - fatality rate also was low in china, with 3 patients dying of the 1,149 reported with ev71 infection. therefore, the virulence of the c4a genotype may be milder than that of other genotypes. possible explanations include the transient presence of the virus in csf, a lower viral load in csf, and use of an ev pcr assay that had not been optimized to detect ev71. in our population, the csf profile of ev71 infection appeared to be broadly similar to that of other cases of viral encephalitis or meningitis. thus, analyzing the csf of patients with suspected ev71 infection may provide minimal information. several studies have shown that ev71 infection rate was most common during the warmer season. in our study, the seasonality of ev71 infection initially was similar to that of previous reports. however, the prevalence of ev71 infection decreased drastically in august, the warmest month in south korea. a possible reason for this difference could be that in 2009, influenza pandemic (h1n1) 2009 affected south korea; with the first death caused by it in south korea reported in august. as a consequence, personal hygiene practices, such as handwashing and covering one s cough or sneeze, were emphasized to prevent virus spread. considering the transmission route of ev71 infection, the emphasis on personal hygiene may thus have hindered the spread of ev71, as well as of the influenza virus. we report 94 cases of pcr - confirmed ev71 infection with cns involvement, including 2 deaths, and provide additional clinical and virologic information about ev71. we confirmed that ev71 commonly involved the brainstem and cerebellum, and therefore ataxia is not uncommon in ev71 infection with cns involvement. in addition, our study supports the hypothesis that the severity of and case - fatality rates for ev71 infection may differ by genotype or subgenotype of ev71.

we assessed neurologic sequelae associated with an enterovirus 71 (ev71) outbreak in south korea during 2009. four of 94 patients had high signal intensities at brainstem or cerebellum on magnetic resonance imaging. two patients died of cardiopulmonary collapse; 2 had severe neurologic sequelae. severity and case - fatality rates may differ by ev71 genotype or subgenotype.

cronkhite - canada syndrome (ccs) is a rare nonfamilial polyposis syndrome characterized by two kinds of marked epithelial disturbances. the first is epidermal manifestations including alopecia, onychodystrophy, and hyperpigmentation, and the second is gastrointestinal tract expressions involving hamartomatous polyps of the juvenile type. usually, ccs colon polyp has been known as a benign neoplasm, but the possibility of serrated adenoma associated with malignant neoplasm was reported in some japanese cases. in south korea, 13 ccs cases have been studied until now, yet there was no case accompanied by the colon cancer. the authors diagnosed a ccs patient who was accompanied with multiple polyps in the stomach, the small bowel and the whole colon. throughout the endoscopic colon polypecotmy, adenocarcinoma in situ along with tubular adenoma and serrated adenoma this is the first case of ccs associated with colon cancer and serrated adenoma in south korea, so we report here with a literature review. a 72-year - old male complained of chronic diarrhea and weight loss of 10 kg in 1 month. he took a colonoscopy examination at a private clinic showing multiple colonic polyps of varying sizes. there were no abnormalities in his past medical and family history such as gastrointestinal polyposis or colorectal cancer. he had been drinking about 1.5 bottles of soju (korean liquor) every day for 20 years. he was showing chronic signs of illness, but his vital signs were relatively stable. he was 164.3 cm tall and weighed 47 kg (body mass index, 17.4). his physical examination revealed mild hair loss and hair pull test showed that more than 10 hairs were pulled out (fig . also, all fingernails of both hands were dry, cracked, and transformed ( fig . a peripheral blood test showed a hemoglobin level of 13.1 g / dl, hematocrit 38.2%, and evidence of megalocytes with mean corpuscular volume of 105.2 fl, mean corpuscular hemoglobin of 36.1 pg, and mean corpuscular hemoglobin concentration of 34.3% . iu / l, an alanine aminotransferase of 24 iu / l, total protein level of 5.5 g / dl, albumin level of 3.4 g / dl, total bilirubin level of 0.6 mg / dl, calcium level of 8.6 mg / dl, blood urea nitrogen ( bun) level of 10 mg / dl, creatinine level of 0.8 mg / dl, na / k / cl / co of 139/4.3/104/27 meq / l, and serum carcinoembryonic antigen level of 5.8 ng / ml. the esophagus was normal in gastroscopy, but several hundred strawberry - like polyps of varying sizes (0.5 to 2.5 cm) were observed in the stomach (fig . the number and sizes of the polyps increased in the distal stomach than in the proximal stomach . rapid urease test ( campylobacter - like organism, clo test) showed positive and the stool helicobacter pylori ag were also positive. the duodenum was covered with multiple small hyperemic polypoid lesions and numerous tiny polyps on whole small bowel were also observed by capsule endoscopy, but these were not confirmed histologically (fig . thousands of little grape - like polyps that were less than 10 mm in size and some colon polyps of more than 1 cm in size were observed during colonoscopy . small scale - like hyperemic mucosa under the size of 1 mm was observed without normal mucosa in the terminal ileum . these colon polyps were resected by endoscopic method, and the pathologic confirmed colon cancer ( adenocarcinoma in situ) (fig . most of them were inflammatory polyps containing lamina propria, and proliferated tortuous and cystic dilated glands were observed ( fig . however, adenocarcinoma in situ with low grade tubular adenomas was confirmed in one of them ( fig . 3b, c). also, another polyp showed serrated architecture and cytological dysplasia of the crypts, corresponding to serrated adenoma (fig . prednisolone of 40 mg / day was administered, and the dosage was reduced by 5 mg every week . h. pylori eradication ( proton pump inhibitor, amoxicillin, and metronidazole combination therapy for 2 weeks) was performed and the follow - up clo test was negative. at 4 months follow - up, although the patient appeared to have improved, hundreds of 0.5 to 2.5 cm - sized hyperplastic gastric polyps were not improved compared to the previous endoscopic findings. in the follow - up colonoscopy, numerous colon polyps were still discovered in the whole colon. additional colon polypectomy was performed and the pathologic confirmed inflammatory polyps and tubular adenoma. since cronkhite and canada described the first two cases in 1955, the number of reported cases has increased to more than 400 over the last decade in the world. the male - to - female ratio was 1.3:1 and 80% of the patients were over 50 years of age at the time of presentation.1 the characteristic two features of ccs are gastrointestinal polyposis and ectodermal changes that consist of alopecia, nail dystrophy, and hyperpigmentation. ccs usually evolves rapidly over several months and mild gastrointestinal and nutritional deficiency symptoms progress to substantial weight loss and general edema. ectodermal changes are usually observed several weeks or months after gastrointestinal symptoms have begun and then two or more of the cutaneous triad appear in most patients.2 until now, etiological studies have been limited because of very low incidence rate. unlike many other polyposis syndromes, familial patterns of inheritance have not been identified in ccs. the japanese author of the largest series described mental stress such as psychological suffering or family problems and physical fatigue as the important precipitating factors for their patients.3 the unique sequential involvement of two epithelial tissues suggests that potentially reversible derangements in epithelial cell - to - cell signaling or maturation may play a pivotal role in initiating the syndrome. although no experimental evidence has validated this hypothesis, the observation that sulindac administration led to regression of ccs polyps may be interpreted as consistent with this mechanism.4 the efficacy of corticosteroids provides the strongest evidence to suggest an inflammatory cause for ccs. a recent provocative report described the infiltration of immunoglobulin g4 (igg4)-producing plasma cells in half of the ccs polyps studied in seven affected individuals. the authors of this report speculated that ccs was an intestinal manifestation of igg4-related autoimmune disease.5 the diagnosis of ccs depends on clinical manifestations. important baseline blood include serum electrolyte (e.g., calcium, magnesium, potassium, and zinc), bun, creatinine, albumin, and total protein levels, as well as the prothrombin time and/or the activated partial thromboplastin time and complete blood count. upper gastrointestinal series along with small bowel follow through is used to evaluate polyps in the stomach and small bowel. wireless capsule endoscopy has recently been used to visualize the abnormal mucosal appearance throughout most of the small bowel. the universal histologic finding is hamartomatous polyps of the juvenile type throughout the gastrointestinal tract without typically involving the esophagus. polyps are noted in the small bowel in approximately one half of all patients, most often in the duodenum and terminal ileum. mucosal changes are characterized by intact surface epithelium, edematous chronically inflamed lamina propria, and proliferated tortuous glands. some of glands are cystically dilated and filled with proteinaceous fluid or inspissated mucus.1 adenomatous changes and carcinoma can occur from hamartomatous polyps in almost 15% of affected patients.6,7 with the increasing observation of gastric and colorectal cancer in patients with ccs, various hypothesizes about this association have been suggested. in literature review , serrated adenoma has been reported in high prevalence in ccs associated with colorectal cancer. a japanese group reported that 40% serrated adenomatous polyps in ccs were detected and it was significantly higher compared with about 1% incidence of non - ccs patient's gastrointestinal polyps. therefore, they proposed the possibility of a serrated adenoma - carcinoma sequence underlying some ccs.7 our present case is a ccs where serrated adenoma was discovered together with colon cancer. so we think that there is a possibility of a serrated adenoma - carcinoma sequence in this ccs patient. when the histological findings of 13 south korean ccs cases were analyzed, most cases had hamartomatous polyps or inflammatory polyps and only one case showed tubular adenoma (table 1).8 - 19 however, in our case, adenocarcinoma in situ accompanying by dysplasia and tubular adenomas were discovered. there was no evidence - based medicine or systematic investigations of medical and surgical interventions for ccs treatment. usually, available management includes nutritional support, administration of zinc, acid suppression, cromolyn sodium, corticosteroid, and eradication of h. pylori. in one case, the successful use of nonsteroidal anti - inflammatory drugs to regress ccs polyps was described,4 but many anecdotal reports support the use of corticosteroid.20 however, the number of data supporting corticosteroid use are insufficient, so it is difficult to accept that as a recommendation. in the 13 ccs cases reported in south korea , small bowel work - up was performed by small bowel series, computed tomography enterography or capsule endoscopy and examination of h. pylori infection was carried out by clo test in only one case. methylprednisolone and prednisolone were administered in four cases (30%) (table 1).8 - 19 in one case, they reported antibiotic treatment (tetracycline) for diarrhea and another case was treated with h. pylori eradication (table 1).8 - 19 in the present case, we tried medical treatment with endoscopic polypectomy. a 40 mg of prednisolone was started and was reduced by 5 mg every week. the prognosis has not been good. a 5-year mortality rate of 55% was reported and malnutrition, hypoalbuminemia, repetitive infection, sepsis, heart failure, and gastrointestinal bleeding were considered as the causes of death.8 there is no guideline for follow - up and surgical treatment. however, we think that a follow - up endoscopic polypectomy can be considered as a follow - up method when there is the possibility of cancer, as in our case.

cronkhite - canada syndrome (ccs) is a rare nonfamilial polyposis syndrome characterized by epithelial disturbances both in the gastrointestinal tract and in the epidermis. the pathologic finding of the polyp is usually a hamartomatous polyp of the juvenile type; however, the possibility of serrated adenoma associated malignant neoplasm was reported in some japanese cases. up till now in south korea, 13 ccs cases have been reported, but there was no case accompanied by the colon cancer. we report the first case of ccs associated with malignant colon polyp and serrated adenoma in korea. a 72-year - old male patient who complained of diarrhea and weight loss was presented with both hands and feet nail dystrophy, hyperpigmentation, and alopecia. endoscopic examination showed numerous hamartomatous polyps from the stomach to the colon. the pathologic confirmed colon cancer and serrated adenoma. helicobacter pylori eradication and prednisolone was used. thus, the authors report this case along with a literature review.

antiretroviral therapy (art) suppresses efficiently the replication of human immunodeficiency virus type 1 (hiv-1) to undetectable levels with standard techniques in most treated patients, but there is still an ongoing low - grade replication in most or all patients. also, immune activation is a central feature of progressive hiv-1 infection , and although the degree of immune activation is decreased during art, it is not normalized. this is especially important since studies suggest that the remaining immune activation may cause organ damage, for example, an increased risk for cardiovascular diseases and possibly neurocognitive dysfunction. the gastrointestinal (gi) immune system seems to play a central role in the pathogenesis of immune activation. the early dramatic depletion of cd4 + t cells from the gut mucosa may drive immune activation, as this mucosal immune damage impairs the normal barrier function and allows increased translocation of bacterial products from the gut lumen into the circulation. we and others have shown that microbial translocation is present in hiv-1 infection through increased plasma lps levels in subjects with progressive disease and that the levels are decreased by art. furthermore, we and others have implied that the alarmin high - mobility group binding 1 protein (hmgb1) modulates hiv-1 replication in vitro and contributes to the activation of immune system. thus, plasma hmgb1 levels are elevated in hiv-1-infected patients and reduced with effective art. hmgb1 is released from damaged or necrotic cells to the extracellular milieu, in which it may act as a potent proinflammatory marker by stimulating cytokine expression in monocytes and endothelial cells. hmgb1 per se does not seem to have a pro - inflammatory activity but has a high affinity to form complexes with other molecules such as lps and cpg - dna. these complexes are likely to bind to various receptors, including tlr4 and tlr9, and promote a large variety of inflammatory and immunological responses. the aim of our study was to explore whether complexes of hmgb1 and tlr ligands, such as flagellin, could synergistically induce hiv-1 replication in a promonocytic cell line. all research involving human participants has been conducted according to the principles expressed in the declaration of helsinki. patients gave their informed written consent and the study protocol was approved by the regional ethics committee in stockholm, sweden (dnr 2005/3:10). lipopolysaccharide (lps) and (phorbol-12-myristate-13-acetate) pma were obtained from sigma (st . louise, mo, usa), il-1 from r&d systems (minneapolis, mn, usa), and cpg - odn type b (odn2006), purified flagellin (s.typhimurium), and anti - flagellin (flic) antibodies from invivogen (san diego, ca, usa and abcam, cambridge, uk). recombinant hmgb1 (hm-116) was purchased from hmgbiotech (milan, italy) or from r&d systems (minneapolis, mn, usa). we also used recombinant hmgb1 that was a kind gift from professor helena erlandsson - harris cmm / ki, stockholm. u1 cells, a subclone derived from u937 cells, were obtained through the aids research and reference reagent program (niaid, nih). the u1 cells are chronically infected with hiv-1 and are characterized by low constitutive levels of virus expression that can be upregulated by several cytokines and phorbol esters. the cells were maintained in rpmi medium (gibco) supplemented with 10% fetal calf serum, glutamine, and antibiotics. cells were seeded at 200 000 cells / ml in 96-well plates and complexes / tlr - ligands / controls were added and incubated for 48 or 72 hours. patients (n = 51) given art, followed at the department of infectious diseases, karolinska university hospital, stockholm, and 19 healthy controls were included. patients' recruitment was based on sample availability as well as virologic response after 2 years of art. thirty - three individuals had undetectable viral load and 18 had detectable viraemia (nonresponders) after 2 years of treatment. the age and sex distribution of the patients and controls was similar (median age 38 years, 52% women). briefly, necrosis was induced in peripheral blood mononuclear cells (pbmcs) from healthy donors (30 10 cells / ml) by exposing the cells to six cycles of freezing and thawing. cell debris was removed by centrifugation and the supernatant was passed through a 0.2 m membrane and collected. the concentration of hmgb1 in necrotic extracts was 40 g / ml, as estimated by immunoblot (data not shown). furthermore, 250 l of necrotic extract was incubated with polyclonal anti - hmgb-1 antibodies (abs) from abcam (cambridge, uk). immune complexes were removed by adding 25 l of sepharose a / g to the extract, incubated for 1.5 hours at 4c, and centrifuged. the supernatant was collected and the procedure was repeated again with 25 l sepharose for 1 hour at 4c. necrotic extract or hmgb1was mixed with the tlr - ligands, lps, cpg - odn, il-1, and flagellin in pbs in different concentrations and incubated for 16 hours at 4c. the suboptimal stimulatory concentrations (capable to trigger hiv replication from u1 cells) of necrotic extract as well as lps, flagellin, cpg - odn, and il-1 were estimated in a series of experiments (data not included). complexes were also mixed and denatured by heating at 95c for five minutes to verify the stimulatory effect of complex formation on u1 cells. equal volumes of necrotic cell extracts, hmgb1-depleted necrotic cell extracts, as well as recombinant hmgb1 proteins were resolved on 1020% tris / glycine gel and transferred onto nitrocellulose membrane (invitrogen, carlsbad, usa). the membranes were then incubated overnight with anti - hmgb1 abs at 1: 2000 dilution. the following day, the membranes were incubated 1 h with horseradish - peroxidase (hrp-) conjugated secondary antibody (ge, healthcare), raised against rabbit igg at 1: 10,000 dilution. approximately 1.88 g of recombinant flagellin was twofold serially diluted (4 series) and subjected to gel electrophoresis on 1020% precasted sds - page gel (invitrogen) in tris / glycine / sds buffer. similarly, bacterial extracts of flagellated e. coli strain o126:h2 and aflagellate e. coli o21:h-(ccug catalog number 11425 and 11326, kind gift from professor andrej weintraub, clinical microbiology / ki, stockholm) that were prepared freshly from an overnight inoculation were also resolved on sds - page gel as described previously. the proteins were then electroblotted onto iblot gel transfer stacks nitrocellulose membranes, using the iblot dry blotting system (invitrogen), as recommended by the manufacturer. after blocking the nitrocellulose membranes for 1 hour in blocking buffer (pbs supplemented with 0.05% tween and containing 10% nonfat milk), the blots were probed with primary antibodies overnight at 4c with a slow agitation. as primary antibody, serum from hiv-1-infected or control subjects diluted 1: 1000, monoclonal, or polyclonal anti - flagellin abs was used. the following day, the membranes were washed with pbs containing 0.05% (vol / vol) tween and bound antibodies were then detected by using hrp - conjugated secondary abs (pierce) against human igg in 1: 10,000 dilution. protein bands were visualized by chemiluminescence (thermo scientific). to confirm the protein bands, two immunoblotted membranes from hiv-1-infected patients and control subjects were stripped off and reprobed with mouse monoclonal antibody directed against flagellin. bound antibodies were then detected by using an hrp - conjugated secondary antibody, raised against mouse (dako ; 1 : 4.000). antibody titers against flagellin, measles, and total igg levels were assessed by elisa. an in - house anti - flagellin - specific igg elisa was developed using purified flagellin monomers from s. typhimurium (invivogen). it has been previously shown that human sera have a similar recognition pattern of flagellin monomers whether isolated from flagellated e. coli or s. typhimurium. briefly , microwell plates (mwp) were coated overnight with purified flagellin from s. typhimurium (25 ng / well). the following day, plasma samples from hiv-1-infected and control subjects diluted 1: 1000 were applied to wells coated with flagellin. after incubation and washing, the mwps were incubated with hrp - conjuggated anti - human igg. for total igg elisa, the enzygnost measeles virus igg elisa kit (behring, germany) was utilized for quantification of antimeasles antibodies. plasma hiv-1 rna levels (cobas amplicor test roche molecular systems ; usa ; detection limit 40 copies / ml) and t - cell counts (flow cytometry) were evaluated as part of clinical routine. supernatants were collected at indicated time points and tested for the presence of hiv p24 antigen with architect i2000 hiv-1 ag / ab combo detection system (abbott diagnostics, abbott park, il, usa). the p24 concentration was calculated based on the several standard dilutions of p24 protein included in each run. differences between groups were analysed with the mann - whitney u - test, and intragroup changes from baseline to the end of the study were evaluated by wilcoxon test. jonckheere - terpstra test was used for trend analyses and correlation analyses were performed using the spearman method. the statistical analyses were performed with spss software, version 15.0 (spss inc, chicago, usa). to determine the impact of an endogenous signal, associated with cell injury, on hiv-1 replication, we generated soluble necrotic extracts from healthy donors pbmcs. additionally hmgb1-depleted extracts were obtained by immune depletion utilizing specific anti - hmgb1 antibodies (figure 1(a) ). in the initial experiment, the u1 cells were exposed to necrotic extract, hmgb1-depleted extract, and pma, respectively. the hiv p24 antigen concentration in the cell supernatants was measured after 72 hours (figure 1(b) ). the levels of viral replication were approximately 2-fold higher after stimulation by necrotic extract compared to the mock cells (p = 0.002). the stimulation with pma gave a 10-fold higher viral replication than stimulation with necrotic extract. notably, addition of necrotic extract depleted of hmgb1 did not in an increase of viral replication, as compared to the controls, suggesting that hmgb1 crucially contributes to the stimulatory effect of the necrotic extract. thereafter, we stimulated the u1 cells with necrotic extract, tlr ligands (lps, flagellin, cpg - odn), and il-1 alone or with the complexes of necrotic extract and the tlr ligands or il-1. notably, stimulation with all the tlr ligands, in combination with necrotic extract, ed in a higher viral replication than stimulation with necrotic extract or tlr ligands alone (figure 2). hence, stimulation with lps, cpg - odn and il-1 in complexes with necrotic extract ed in a 1.52-fold - increased viral replication compared to each component alone, whereas flagellin in combination with necrotic extract ed in a 7-fold increased replication compared to flagellin alone and a 13-fold - increased replication compared to necrotic extract alone. the preheating of complexes prior incubation with cells ed in abrogation of stimulatory signal, implying that the active compound relies on intact protein structure (data not included). in order to explore if hmgb1 could mimic the synergistic effects of necrotic extract - tlr ligands, we challenged the u1 cells with complexes consisting of hmgb1 and bacterial substances. indeed, stimulation with microbial products (lps, flagellin, or cpg - odn) in combination with hmgb1 ed in a higher viral replication than stimulation with hmgb1 or tlr ligands alone (figures 3(a)3(c) ). stimulation with lps, flagellin, and cpg - odn in combination with hmgb1 ed in a 1.52-fold - increased viral replication compared to each component alone, although the stimulatory effect was not as prominent as with tlr ligands in combination with necrotic extract. it is known that immune response to flagellin is mediated by tlr5. to investigate whether anti - tlr5 antibodies could block the inducing effects of flagellin, we first preincubated u1 cells with anti - tlr5 antibodies and subsequently added the necrotic extract complexed with flagellin. flagellin in combination with hmgb1-depleted extract gave a 3-fold - increased viral replication compared to depleted extract alone, whereas flagellin in combination with necrotic extract gave a 4-fold increase compared to necrotic extract alone (figure 3(d) ). preincubation of u1 cells with anti - tlr-5 antibodies before addition of necrotic extract - flagellin complexes ed in a dose - dependent inhibition of this stimulatory effect (p for trend < 0.001). encouraged by the in vitro data we aimed to evaluate whether flagellin is a potentially important antigen in vivo during hiv-1 infection. therefore, serum samples from hiv-1-infected patients and control subjects were used to measure the level of flagellin - specific antibodies by western blot analysis. when diluted 1: 1000, all of the sera samples from the hiv-1-infected patients analyzed exhibited easily detectable bands that recognized the first two dilutions of flagellin derived from s. typhimurium (figure 4(a), upper panels ). a relative increase in flagellin - specific igg in hiv patients in contrast, in only one control subject (cs#2) flagellin was detected faintly at the highest dilution (figure 4(a), lower panels ). although semiquantitative analysis of detected bands was not performed, the levels of flagellin - specific igg observed were in all cases strikingly elevated in hiv-1-infected patients. similar pattern of anti - flagellin igg was observed when plasma instead of sera was used. in order to address the specificity of the flagellin igg , we subjected the bacterial lysates from flagellated and aflagellate e. coli to protein separation on the sds - page gel. the western blotting with hiv-1 serum (as a primary antibody) showed similar pattern as when the polyclonal anti - flagellin antibody was used confirming that the specificity of the antibodies was not limited to the recombinant protein (figure 4(b) ). furthermore, we used the anti - flagellin elisa to evaluate the levels of flagellin igg in plasma of hiv-1-infected patients before and after two years of art. at baseline significantly elevated levels of flagellin antibodies were found in hiv-1-infected patients as compared to controls (p < 0.001) (figure 5(a) ). this difference persisted (p < 0.001) when the flagellin antibodies were adjusted to the total igg (figure 5(b) ), suggesting that the elevation of flagellin antibodies was not due to hypergammaglobulinemia. moreover, analysis of antimeasles antibodies in 10 patients with severe immune deficiency (cd4 + t - cell counts 200) supported that the elevation of the flagellin antibodies was not caused by polyclonal activation supplementary table 1 (see supplementary material available online at doi:10.1155/2012/263836). the levels of flagellin igg, total igg, and the ratio flagellin igg / total igg were significantly reduced after two years of art for the whole group (p < 0.001, p = 0.03, and p < 0.001, resp . additionally a significant reduction of flagellin igg levels was observed also when the patients were subdivided into those with successful art and the nonresponders who had remaining low levels of viral replication two years after initiating the art ( p = 0.009 ; p = 0.001, resp .). the total igg levels after art did not decrease in nonresponders as they did in successfully treated patients (p < 0.001) (data not shown). we found no correlation between the levels of flagellin igg and the viral load nor the cd4/cd8 t - cell counts. in contrast, among the subgroup of 42 patients in whom we had earlier analysed hgmb1 and lps in plasma, significant correlations were found between the levels of flagellin igg and lps (r = 0.32 ; p = 0.02) as well as between the flagellin igg / total igg ratio and lps (r = 0.25 ; p = 0.007) (data not shown). microbial translocation has been described in different conditions like inflammatory bowel disease, neutropenia, and chronic viral infections. in hiv-1 infection , the proof for the translocation of bacterial products is based mainly on lps data. however, the original observation that increased lps levels were associated with both activated memory cd8 + t cells and enhanced ifn- levels implies the involvement of other factors. we therefore hypothesized that hmgb1 could be such a link between the microbial products and hyperinflammation. mounting evidence shows that hmgb1 does not act alone but forms stable potent proinflammatory complexes with other molecules, such as bacterial products or single stranded dna. since we have earlier shown that hmgb1 alone activates latent hiv-1 replication in vitro, we decided to expand our analysis to the effect of hgmb1 in complexes with bacterial products. here , we present that hmgb1 in complex with the tlr ligands (lps, cpg - odn, flagellin) and il-1 induce viral replication in a promonocytic cell line, u1 cells. the data obtained with both the hmgb1 derived from necrotic extract as well as recombinant protein yielded similar , although the stimulatory signals associated with necrotic hmgb1 were more potent. this is not surprising as other endogenous danger signals should be anticipated in this process. the reduction of the stimulatory effect by depletion of hgmb1 also supports our hypothesis that hmgb1 is an important component of these complexes. these in vitro findings brought our attention to flagellin as a potent activator of hiv-1 replication alone or in complexes with hmgb1. bacterial flagellins are present in all motile bacteria and play an important role in mediating gut inflammation associated with infection by enteric pathogens or in inflammatory bowel diseases. their proinflammatory activity is exerted mainly through tlr5. it has been recently demonstrated that flagellin is the major antigen activating innate and adaptive immune response in intestinal inflammation observed in crohn's disease. disruption of the intestinal barrier promotes translocation of flagellated commensal bacteria across the epithelium driving the activation of innate immune cells residing in the lamina propria. this phenomenon also in abnormal exposure of immune cells to flagellin, a process that may influence the balance and function of the different t - cell subsets present in the gut - associated immune system (galt) and promote inflammation. the contribution of flagellin to immune activation during hiv-1 infection has been anticipated, but the data are scarce. thus, exposure of pbmcs to flagellin ed in activation of t cells predominantly of central memory and effector memory phenotype. moreover, flagellin is able to induce hiv-1 gene expression in resting memory cd4 + t cells that are considered as a key cellular hiv-1 reservoir in infected individuals. our in vitro and in vivo findings are clearly in line with the hypothesis of an important role of flagellin in hiv-1 pathogenesis. thus, we demonstrated that flagellin complexes are able to significantly stimulate the hiv-1 replication, at least from cells of monocytic origin. furthermore, our finding that elevated levels of anti - flagellin igg are present in hiv-1-infected individuals anticipates that this observation has in vivo implications. hence, we show not only presence of elevated levels of flagellin antibodies before the initiation of art but also a reduction after two years of art suggesting decreased exposure to the antigen probably due to partial restoration of gut - blood barrier. the hypergammaglobulinemia present during the hiv-1 infection can not be solely responsible for the elevation of flagellin igg levels as the normalisation to the igg did not influence the . an elevated adaptive immune response to flagellin has been previously observed in conditions associated with gut barrier dysfunction such as crohn's disease and short bowel syndrome and relates to the severity of crohn's disease. also, kamat et al. reported recently the presence of a subgroup of anti - flagellin antibodies (anti - cbir1) in 4/26 hiv-1-infected patients with cd4 + t - cells counts < 300 cells / ul and high lps levels. the cbir1 flagellin has been identified as an immune dominant flagellin in crohn's disease and linked to clostridia species. interestingly a recently presented work has shown alterations in bacterial composition of microbiota during hiv-1 infection with significantly lower ratio of clostridia taxa in faeces obtained from hiv-1-infected patients as compared to controls. although our assay is based on the recognition of salmonella typhimurium flagellin which spans the two well conserved n- and c - terminal domains of flagellin enabling us the detection of broader range of flagellin antibodies, additional studies are needed to determine the antibody specificity. furthermore, our deserve future studies of adaptive flagellin immune response in a larger cohort of hiv-1-infected individuals. in summary, the novelty of our findings is that the bacterial products and hmgb1 form active complexes which can efficiently not only create a proinflammatory milieu but also directly trigger viral replication in infected cells. this synergistic effect may require lower levels of the interacting substances when present in complexes, as suggested by others. we also report that flagellin has to be considered as a microbial product that can contribute to the immune activation during the hiv-1 infection. the formation of hmgb1/tlr ligand complexes has direct implications on immune activation, particularly in late stage of disease, where cell destruction and necrosis are dominant phenomena due to cd4 + t - cell loss, opportunistic infections, and other pathological conditions.

objective. we hypothesized that hmgb1 in complex with bacterial components, such as flagellin, cpg - odn, and lps, promotes hiv-1 replication. furthermore, we studied the levels of antiflagellin antibodies during hiv-1-infection. methods. chronically hiv-1-infected u1 cells were stimulated with necrotic extract / recombinant hmgb1 in complex with tlr ligands or alone. hiv-1 replication was estimated by p24 antigen in culture supernatants 4872 hours after stimulation. the presence of systemic anti - flagellin igg was determined in 51 hiv-1-infected patients and 19 controls by immunoblotting or in - house elisa. . flagellin, lps, and cpg - odn induced stronger hiv-1 replication when incubated together with necrotic extract or recombinant hmgb1 than activation by any of the compounds alone. moreover, the stimulatory effect of necrotic extract was inhibited by depletion of hmgb1. elevated levels of anti - flagellin antibodies were present in plasma from hiv-1-infected patients and significantly decreased during 2 years of antiretroviral therapy. . our findings implicate a possible role of hgmb1-bacterial complexes, as a consequence of microbial translocation and cell necrosis, for immune activation in hiv-1 pathogenesis. we propose that flagellin is an important microbial product, that modulates viral replication and induces adaptive immune responses in vivo.

fragile x syndrome (fxs) is the most common mental disorder caused by a cgg trinucleotide amplification on xq27.3 in the 5 untranslated region of fmr1 gene cloned and named in 1991, which suppresses production of fragile x mental retardation protein (fmrp). fmrp is widely expressed in neuron and glia in brain and acts as an interactor, regulating mrna shuttling, translational control, and synaptic plasticity in copious encephalic regions which are responsible for cognition, emotions, and memory. in fxs individuals, compared to attention deficits and hyperactivity which were common in childhood but declined remarkably throughout adolescence and adult years, the morbidity of anxiety remains high with impaired ability of information process. it is consistent with the common viewpoint that anxiety is a long - lasting response to danger signals that are either from immediate circumstances or from vague indications of ill - defined events. in short, anxiety is derived from anomalous regulation of fear. in addition, as one major mood disorder associated with fxs, anxiety occurs with premutation (alleles between 55 and 200 cgg repeats) or full mutation (alleles that exceed 200 cgg repeats) in both genders and affects limbic system and neocortex. specifically, limbic system and paralimbic system participate in formation and maintenance of anxiety associated with fxs which mainly involve amygdala, prefrontal cortex (pfc), insula, cingulate cortex, temporal cortex, and hippocampus, etc.. insula is found to be the location where 5-httlpr (5-hydroxytryptamine transporter linked polymorphic region) might cause anxiety. the hypofunction of prefrontal cortex and anterior cingulate cortex supports the top - down control mechanisms of anxiety process in affected individuals. the frontostriatal deficits and the dysfunction of the frontoparietal network are proposed to be critical for anxiety processing of external stimuli, etc.. besides the complicated neural network with abnormal expression of fmrp, the dysgenesis of dendritic spine also significantly influences the synaptic plasticity which accounts for anxiety disorders associated with the development of fxs. and dysfunctional circuits could lead to abnormal spines and vice versa, so it is difficult to figure out which one comes first. because of intricate involvement of proteins regulated by fmrp in synaptic plasticity through maintenance of spine shape and dynamics, the two defects are arguably inseparable. in the present review , we explored how structure and function coordinately work to promote the anxiety process in fxs and emphasized the selective and monolithic modulation model of the progression. plasticity is considered as a critical process in pain, learning, memory, emotion, cognition, and so on. substantial evidences have demonstrated that structural changes coordinated with functional changes induce synaptic plasticity, in which ltp and ltd are reciprocally modified by spine density and morphology in fmr1-knockout (ko) mice. taken together, change of plasticity induced by defects in spine morphology or neural circuits is significantly involved in the process of anxiety in fxs. on the one hand, it is a significant symbol that the affected individuals have increased quantity of longer dendritic spines. also, it has been reported that spines were altered in very young fmr1-ko mice, although spine alterations disappear in adolescent mice and reappear in adult so far, these observations were acquired in traditional areas, while high - level cognitive regions had not received enough attention. comparative study in more brain regions of fmr1-ko mice fmrp plays a vital role in activity - dependent synapse elimination, as well as in spine stabilization, to increase the cell - autonomous spine density. for example, in mouse model of fxs, spine density and morphology are altered in an age-, region-, and cell type - specific pattern (table 1). on the other hand, it is widely acknowledged that ltp and ltd are molecular mechanisms underlying cognition and emotion. previous studies showed enhanced metabotropic glutamate receptor- (mglur-) ltd and impaired cortical ltp in fmr1-ko mice. group i mglurs are linked to translational activation in neurons and stimulate rapid synthesis of fmrp at synapses. due to the link of mglurs with fmrp, mglur - dependent ltd was enhanced in hippocampus and cerebellum in fmr1-ko mice. moreover, cortical ltp is known to be impaired in learning and fear / anxiety memory, including both mglur - dependent and nmdar - dependent ltp. and it is expectable that deficits in mglur - dependent ltp and non - mglur - dependent ltp in the anterior cingulate cortex and amygdale were correlated with anxiety - like behaviors of fxs. interestingly, the activation of mglur5 is involved in the late phase ltp (l - ltp) and synaptic depotentiation. this plasticity induced by mglurs is not simply the alteration of synaptic strength but the change of inducibility of later synaptic plasticity, which suggests that the activation of mglur5 regulates the transport and function of nmda receptor. furthermore, fmrp induced by activation of mglur5 could be the common molecular mechanism of these phenomena by regulation of transport of nmda and ampa receptors. these suggest that the lack of fmrp may impair ltp and attenuate cortical network recruitment. together , the loss of fmrp may participate in cortical ltp deficits via ampa receptors internalization at postsynaptic neuron. taken together, dysgenesis of dendritic spine and deficits of synaptic plasticity in the formation of anxiety in fxs mouse models. spine density and morphology have an alteration by an age-, region-, and cell type - specific manner. also, the defect in spine maturation and pruning is correlated to dysfunction of neural circuits and deficits of synaptic plasticity. on the other hand, thus, the ampa receptor internalization, the inheritable rescue of mglur - ltd, and the rescue of the specific dendritic spine might share the common mechanistic basis. in brief, dendritic spine dysgenesis and defects in synaptic plasticity promote each other in a structure - function interdependent manner. in human genetics, the cgg expansion in the promoter region, which includes the cpg island of fmr1 gene, is hypermethylated and provokes the silencing of the transcription of the fmr1 gene, leading to the absence of fmrp, while in the ko mouse model, it is the classical gene knockout effects that in the silence of fmr1 gene and the loss of fmrp, synaptic function, and plasticity. what is more, recent laboratory studies have provided increasing evidence for the role of fmrp in translational suppression via ribosomal stalling and microrna. and more evidence about the characteristic of fmrp, a polyribosome - associated rna - binding protein, reveals more profound mechanisms relevant to abnormal synaptic plasticity. furthermore, fmrp does not regulate single synapse; instead, it regulates cell - to - cell connectivity. specifically, at synapses involved in specific situation, two neurons are simultaneously removed, retained, or matured, where fmrp ultimately cause dysfunctional consequences. there are two main theories illustrating the interaction of fmrp and neuronal activity in the cortical circuits. the anomalous functions of mglurs - dependent synaptic plasticity have been observed in hippocampus of fmr1-ko mice. activity - dependent synthesis of fmrp in enduring forms of synaptic plasticity may be induced via exaggerated mglur - ltd in hippocampal neurons, while the initiation of long - term potentiation (ltp) is a qualitatively different functional consequence of group i mglur - stimulated protein synthesis at the synapses of hippocampus where ltd can be induced. besides, the mglur theory proposes that stimulation of group i mglur induces local mrna translation, ing in novel protein synthesis that subsequently enhances the internalization of ampa receptors. this model predicts that, in absence of fmrp, the increased translation of a subset of mrnas disturbs receptor internalization dynamics and then exaggerates internalization of ampa receptors and weakens the synapse. what is more, independent protein synthesis mglur - ltd in fmr1-ko mice suggests that in absence of fmrp, proteins that are significant for the maintenance of mglur - ltd are already largely present at the synapses. overall, the mglur theory presents a well - defined mechanism of anxiety in fxs by all accounts: higher density of spines and more immature spines which lead to deficits and anxiety - like behavioral phenotypes in fxs. the mglur - ltd theory was considered as the main theory of psychological symptoms associated with fxs. moreover, the mglur theory has directed research towards the preclinical mechanisms underlying fxs and led to the effective novel therapeutic strategies. from the further intracellular perspective , the phosphorylation of fmrp influences the translation of target mrnas because unphosphorylated fmrp is associated with actively translating polyribosomes while phosphorylated fmrp is associated with inactive polyribosomes. by and large, there are three signaling pathways downstream of mglur5 affecting translation: the pi3k - mtor, the mek - erk - mnk1, and the camkiv - creb pathways. activation of mammalian mtor cascade in the phosphorylation of protein phosphatase 2a (pp2a) and s6 kinase and mrna translation rapidly. besides, a thesis reported enhanced ras - pi3k signaling input induced by the activation of glua1 enriches the mtor signaling pathway. thus, the overactivated mtor signaling pathway in the hippocampus might play a prominent role in fxs. however, no analogy is reported to compare the relationship between nmda and mtor signaling. also, it has been demonstrated that camp responsive element - binding protein (creb) contributed to the regulation of fmrp by group i mglurs and was closely linked to anxiety. therefore, the main three signaling pathways work together downstream of mglur5 to influence the protein synthesis, synaptic plasticity, and anxiety - like behaviors (figure 1). what is more, the camp theory was proposed as a supplement to mglur theory. apparently it is a potential shortcoming in the context of the mglur theory; ca and pkc are both part of the gq cascade, which are able to regulate certain ac isozymes and disrupt normal function. also, there are evidences that cyclic amp and mglur interact with each other because g protein has the potential to act through a network of multiple overlapping messengers. furthermore, the fact that mglur inhibitors rescue camp deficits in fxs and presumably downstream of excessive mglur activity supports the idea that the two theories operate in series. importantly, fmrp is broadly expressed in gabaergic neurons, indicating that it is involved in normal interneuron maturation and function modulation. indeed, it is significant that gaba can modulate neurotransmitter release in an autocrine or paracrine fashion, via mechanisms at presynaptic gabaa and gabab receptors. for example, on the presynaptic side, where fmrp is also expressed , the expression of gaba - synthesizing enzyme gad in fmr1-ko mice is found increased or decreased , with the change relying on the brain region examined. in summary, gabaergic signaling is essential for regulating neuronal migration, maturation, and circuit formation. and defects in the gabaergic system are, therefore, likely to have profound effects on neuronal development and circuit function in fxs. currently, a better understanding of early developmental alterations in gabaergic system in fxs would be reckoned as the crucial insight into the nature of the fxs brain, as well as valuable information about key pharmacological targets. furthermore, in mature neurons, the ionotropic gabaa receptors mediate postsynaptic hyperpolarization via intracellular cl influx, while gabab receptors activation likewise hyperpolarizes the postsynaptic membrane by activating g - protein - coupled inwardly rectifying k channels. in addition to its role as a postsynaptic inhibitory neurotransmitter, distinct mechanisms modulate neurotransmitter release at presynaptic gabaa and gabab receptors. meanwhile, over behavioral effects, gabaa receptors seem to affect more short - term plasticity, seizures, learning and memory deficits, and poor motor skills on a repetitive task and hyperactivity features. and for instance, papers have reported stx209, a gabab agonist, might improve neurobehavioral function and satisfying effects were obtained in a phase 2 trial. it also worth noting that the role of gaba in the developing cns is dynamic and variable between brain regions. therefore, the same gabaergic effectors that helped adult patients could have adverse effects in developing individuals based on the function of gaba in particular brain regions at specific developmental time periods. overall, both abnormal mglurs and gaba synaptic plasticity play significant synergistic roles in the formation of anxiety. the gabab receptor may serve as the functional link between both pathways, as this metabotropic receptor regulates glutamate release at glutamatergic synapses. in fxs patients, a reduced release of gaba from the gabaergic terminals to the presynaptic gabab receptors may induce a reduced inhibition of neurotransmitter spillover, which in turn activates mglur signaling. activation of group i mglurs enables mobilization of endocannabinoids in the postsynaptic neuron and negatively modulates gaba release through a mechanism known as depolarization - induced suppression of inhibition (dsi) (figure 2). these mechanisms require increased neuronal activity, which exists in brain circuitry of fmr1-ko mice. therefore, in consideration of endocannabinoid mobilization in the fxs, the loss of fmrp may selectively affect specific inhibitory circuits. in the developing and mature brain , it is critical for cortical excitatory neurons to be proper synchronized at behaviorally relevant frequencies. and thus, alteration of mglur signaling in this specific type of interneuron is likely to have wide - reaching effects in developing and mature cortical networks and needs to be further explored. how ltp deficits and enhanced ltd are temporally and spatially coordinated with each other in different regions remains to be determined. from the intrinsic modulation perspective , it has been showed that fmrp would suppress the translation stimulated by neuronal activity and generate neuronal feedback responses to activity at the neuron level. adding to suppression of translation by mirna and stalled polyribosomes, synaptic regulation, which involves glutamate receptor signaling, gaba receptor signaling in neurons, and intracellular pka, pkc, camp, and pi3k signaling pathways, also implicates fmrp. on the one hand, mglur and nmda subtypes, as fmrp targets, have diverging structure and features, determining diverse downstream signaling targets, as described before. besides, the initial role of fmrp associated with its transcripts targets needs more attention. from stimulated receptors on neurons to intracellular signaling, it could be conclusive that fmrp selectively regulates the expression of components of the erk and mtor signal transduction pathways in different brain areas. for example, activating glua1 in the downstream ras - pi3k signaling in hippocampus which shares ras protein with mtor signaling. and these signalings convert receptor activity into translational output. taken together, it seems that fmrp associated with its target transcripts directly regulates translational control of the pre- and postsynaptic proteome and synaptic plasticity in different brain areas. considering the primary and secondary procedures, the process should be examined at different times of the nucleus modulation, mrna, or proteome life - cycle. previously, it has been proposed that rna interference and stalled polyribosomes implicate fmrp. as for now, mirna family, including mir-125b, mir-132, and mir-196a, were found to be relevant to the regulation. furthermore, using moloney leukemia virus (mov10) to unfold the structured argonaute 2 (ago2) through rnai and adenosine - to - inosine rna - editing proposed novel perspectives to explore the widely unknown target rnas and associated regulation pathways. meanwhile, recently, a sensational work reported that cdh1-anaphase - promoting complex (apc) and cdc20-anaphase - promoting complex control the morphogenesis of axons and dendrites and synaptic plasticity. cdh1-apc and fmrp are components of a novel ubiquitin signaling pathway that regulates mglur - ltd in brain in terms of cell cleavage or neuronal development. together, an original work reported that the drosophila fragile x homolog (dfmr1) biochemically interacted with the adenosine - to - inosine rna - editing enzyme dadar. and all this work may propose an original view of nucleus modulation of fmrp and consummate the cognition of fmrp's interactor role on dna, mrna, and stalled polyribosomes levels. therefore, we can conclude that the cleavage and differentiation of neuron, involving gene editing, rnai, and stalled polyribosomes are integrated and implicated patterns. and just these manners interfere with the diverse synaptic plasticity between hippocampus and other regions. generally, anxiety is caused not only by connate factors but also by self - regulation of brain homeostasis. during propofol sedation , fxs subjects have significantly decreased the rates of cerebral protein synthesis (rcps) in brain as a whole, cerebellum, and parts of cortex, which also suggests changes in synaptic signaling can balance increased rates of cerebral protein synthesis (rcps) in fxs. also, considerable laboratory research has accumulated copious evidences based on the changes of synaptic plasticity associated with anxiety. however, there also exist contradictory and promising fields to explore: while further data linking these morphological changes to the functional modifications underlying anxiety process were relatively lacking in fxs, a potential entry point to it is the bdnf - trkb signaling. also, given the respective importance of ampars and the rho gtpases in functional and morphological plasticity, the exploration of interactions between these two signaling pathways may provide a mechanism to illustrate these changes.short-term plasticity (stp) is widely believed to play a key role in synaptic information transmission by optimizing the neural output in response to specific patterns of neuronal activity. although it was identified that presynaptic actions in hippocampus are mediated by the large conductance ca activated k (bk) channel together with the interaction of fmrp, further study is needed.in recent years, the development of ipscs (induced pluripotent stem cells) technology is emerging flourishingly. a typical example is that generation of naive / ground state fxs - ips cells with reactivated fmr1 successfully figure out a mechanism of transcriptional silencing. in this mechanism, fmrp might direct binding the rna - induced silencing complex (risc) on the fmr1 transcript and lead to production of 2226 nt cgg fragments. and then it facilitates fmr1 methylation and silencing by directing histone modifying proteins to the locus.proteins play a complicated role in organisms extensively. lately, a sensational study published in science firstly reported that rqc2 can promote alanine and threonine synthesizing uncompleted protein without the manipulation of dna and mrna. meanwhile, this peculiar phenomenon may also occur in fxs because of its specific stalled polyribosomes. or maybe some special proteins having homogeneous functions remain to be explored. while further data linking these morphological changes to the functional modifications underlying anxiety process were relatively lacking in fxs, a potential entry point to it also, given the respective importance of ampars and the rho gtpases in functional and morphological plasticity, the exploration of interactions between these two signaling pathways may provide a mechanism to illustrate these changes. short - term plasticity (stp) is widely believed to play a key role in synaptic information transmission by optimizing the neural output in response to specific patterns of neuronal activity. although it was identified that presynaptic actions in hippocampus are mediated by the large conductance ca activated k (bk) channel together with the interaction of fmrp, further study is needed. in recent years, the development of ipscs (induced pluripotent stem cells) technology is emerging flourishingly. a typical example is that generation of naive / ground state fxs - ips cells with reactivated fmr1 successfully figure out a mechanism of transcriptional silencing. in this mechanism, fmrp might direct binding the rna - induced silencing complex (risc) on the fmr1 transcript and lead to production of 2226 nt cgg fragments. and then it facilitates fmr1 methylation and silencing by directing histone modifying proteins to the locus. proteins play a complicated role in organisms extensively. lately, a sensational study published in science firstly reported that rqc2 can promote alanine and threonine synthesizing uncompleted protein without the manipulation of dna and mrna. meanwhile, this peculiar phenomenon may also occur in fxs because of its specific stalled polyribosomes. or maybe some special proteins having homogeneous functions remain to be explored. synaptic plasticity reveals flexibility and codes capacity of neuronal networks. in fxs, the absence of fmrp perturbs the balance in array of diverse plastic mechanisms of synaptic plasticity in a developmental and regional dependent way. and along anxiety process, a very close structure - function relationship exists between spines and neuronal activity: synaptic strength, synaptic forms of plasticity involved in learning and memory, and activity - dependent plasticity. from the morphology and structural perspectives, increased density of longer immature spines may induce the synaptic plasticity and may from the loss of fmrp in multiple interlaced brain areas. on the point of molecular mechanisms underlying alterations of synaptic plasticity, the proteins encoded by fmrp target mrnas indicate a high level of control over the balance of activity - dependent translation in synaptic plasticity. first, mglur and nmdar - dependent synaptic plasticity are altered in fxs mouse models. specifically, this dysregulation of processes involves downstream of gp i mglur signaling and ampa receptor internalization, gaba release, and regulation of mglurs and gaba via endocannabinoid. second, fmrp regulates the expression of components of the erk and mtor signal transduction pathways selectively, but not in only one way (table 2). finally, gene editing, endogenous rnai, and stalled polyribosomes may influence the internal regulation of synaptic plasticity. or just this integrated regulation, not single targets, facilitates the pathological progression of plasticity and anxiety - like behavior in fxs.

fragile x syndrome (fxs) is an inheritable neuropsychological disease caused by expansion of the cgg trinucleotide repeat affecting the fmr1 gene on x chromosome, ing in silence of the fmr1 gene and failed expression of fmrp. patients with fxs suffer from cognitive impairment, sensory integration deficits, learning disability, anxiety, autistic traits, and so forth. specifically, the morbidity of anxiety in fxs individuals remains high from childhood to adulthood. by and large, it is common that the change of brain plasticity plays a key role in the progression of disease. but for now, most studies excessively emphasized the one - sided factor on the change of synaptic plasticity participating in the generation of anxiety during the development of fxs. here we proposed an integrated concept to acquire better recognition about the details of this process.

a single examination does not fulfill all the functions of assessment, such as assessing knowledge, comprehension, skills, motivation, and feedback. written examinations (essays and multiple choices) test cognitive knowledge, which is only one aspect of the competency. structuring of questions and assessment through highlighting on objectivity has been emphasized and gained importance in the practical evaluation. the objective structured practical examination (ospe) is now an accepted tool in the assessment of practical skills in both pre- and para - clinical subjects. however, there are no strict or limiting guidelines on the types of scenario that are used in the ospe examinations. in the uk, the usa, canada, and indeed most reputable colleges of medicine, the ospe is the standard mode of assessment of competency, clinical skills, and counseling sessions satisfactorily complementing cognitive knowledge testing in essay writing and objective examination. several universities adopted a similar pattern of practical evaluation, which is un - uniform and largely subjective. biochemistry departments in many medical schools have been using the ospe as an assessment method for assessing students performance in laboratory exercises. the marks awarded generally reflect only the global performance of the candidate and are not based on demonstration of individual competencies. in the university examination, there used to be frequent complaints from external examiners insisting that the existing examination pattern is tedious and time - consuming. the ospe is a versatile multipurpose evaluative tool that can be utilized to evaluate students in practical assessment. it is comprised of several stations in which examinees are expected to perform a variety of practical tasks within a specified time period against criteria formulated to the practical skill, thus demonstrating competency of skills and/or attitudes. ospe has been used to evaluate those areas most critical to perform by students, such as the ability to obtain / interpret data, solve the problem, teach, and communicate. any attempt to evaluate these critical areas in the old - fashioned practical examination will seem to be assessing theory rather than simulating practical performance. an earlier innovation in this regard is the objective structured clinical examination (osce) later extended to the ospe described in 1975 and in greater detail in 1979 by harden and his group. these methods with some modifications have stood the test of time and have largely overcome the problems of the conventional clinical / practical examinations mentioned earlier. in view of this , we tried the system of ospe for the assessment of practical in the subject of biochemistry for the first time. if the assessment pattern consists of a variety of methods that demand understanding of the subject matter, this problem can be solved to some extent. to test the earlier observation that a single exam does not fulfill all the functions of assessment, such as assessing knowledge, comprehension and skills, motivation, and providing feedback, we developed an evaluation system. we undertook this study to evaluate whether ospe could be a method of learning and assessment of practical skills in biochemistry and to explore the student, to determine student satisfaction regarding the ospe as a method of assessment of laboratory exercises and to explore the faculty perception of ospe as a learning and assessment tool. the first m.b.b.s students admitted for 2011 - 2012 batch of medical college were the subjects for the study. after successfully completing the syllabus pertaining to the topic on identification of unknown abnormal constituents in urine in practical and unknown abnormal constituents in urine in theory, ospe notification was announced 30 days in advance. this study was undertaken to determine the reliability and student satisfaction regarding the ospe as a method of assessment of laboratory exercises in biochemistry before implementing it in the forthcoming university examination. before administering this tool for evaluation, all the staff members involved in designing and conducting ospe were trained by attending an " workshop on ospe / osce " conducted by medical education unit, medical college. ready - made and peer agreed upon check list formed the basis of assessment in procedure station. structured questions were formed for question stations and key answers for the same were also prepared. since the assessment was being carried out for the first time, the students were oriented toward such a system in advance before administering the tool. each station was designed such that the task could be completed comfortably within 5 min. coefficient of reliability of questions administered was done by calculating cronbach's alpha. a questionnaire on various components of the ospe however, 49 students on an average achieved > 75%, 52 students achieved between 65% and 75%, and 29 students scored between 50% and 65%. average scores of students the mean scores of each station and the score obtained by calculation of cronbach's alpha for testing the internal consistency of the questions administered are depicted in table 1. student feedback analysis of response to various aspects of ospe is depicted in table 2. feedback from faculty on ospe as an evaluation system has been shown in table 3. the mean scores of each station and the score obtained by calculation of cronbach's alpha for testing the internal consistency of the questions administered student feedback analysis of response to various aspects of objective structured practical examination feedback from faculty on objective structured practical examination as an evaluation system feedback on most appreciated aspects about objective structured practical examination by students over the years, increasing experience with the procedure has led to the use of ospe not merely as an evaluation tool but as a teaching method. this has largely been attributable to the feedback that ospe gives both to students and teachers. among 150 students in 1 m.b.b.s batch , four students could not attend due to their personal reasons. of the 146 students, 101 students performance was highly satisfactory, who scored > 65% of marks on an average. however, 16 students did not manage to get even 50% of average marks as their performance was equally poor in both performance and question stations. evaluated marks of question station and check list of procedure station were made available to the students, who appreciated what they achieved and identified and where they need to improve. feedback given by students was constructive and showed high acceptance, which are presented in table 2. ninety - nine percent of students believed that ospe helps them to improve and 81% felt that this type of assessment fits in as both learning and evaluation tools. however, 65% of students expressed that ospe to be introduced partially in the final exams. a vast majority of students enjoyed ospe because of its objectivity, more student assessment in less time, and uniformity. however, 60% of faculty agreed upon the use of ospe in both formative and summative evaluations. the university examination is conducted in both forenoon and afternoon sessions for 6 consecutive days. in the university examination, students used to complain about the irrelevant questions asked by the examiners and also the subjectivity of the examination. they used to complain that the questions asked in the performance exercises varied in difficulty, giving rise to much variation in the scores. here , ospe was adopted with the intention of restricting the examination only in the forenoon sessions, thereby reducing the total time, to make the assessment uniform for all students, and also to reduce the stress of students by making them go through only one round of examination instead of two rounds. the scoring is objective, since standards of competence are pre - set and agreed check lists are used for scoring. a large number of students can be tested within a short time. from the students point of view this study reveals the importance of the role of students in developing new assessment tool. this type of assessment serves as a tool for testing multiple dimensions of student performance because it tests both skills as in performance exercises and knowledge as in ospe. our findings correlate with the earlier findings that multiple choice questions test more of factual recall. nevertheless, if framed properly, multiple choice questions could be used to assess different levels of intellectuality. our agree with the earlier findings that a single type of assessment alone does not meet all the criteria for evaluating student performance. it also helps teachers to think about innovative methods of teaching and evaluation to improve the relevance of biochemistry and to modify question format to improve relevance and comprehension of questions in the succeeding exams. , we found that ospe was more objective, measured practical skills better, and eliminated examiner bias. student feedback reflects that such assessment helps them to improve as it is effective both as teaching and evaluation tools. faculty participated in organizing ospe felt that such exercises can be given frequently for formative evaluation before introducing it in summative evaluation. we have outlined the features of the evaluation system followed in our setup, and based on the feedback, we consider that it would help students to develop different learning skills and make them better learners. experience and experimentation will inevitably in the refinement of the ospe as a tool for learning and evaluation. however, in the current situation, it may not be realistic to expect its inclusion in the formal summative evaluation schedule of universities. however, it is feasible in view of the tremendous advantages that it offers, to include the formative (day to day) assessment of students to improve their clinical competence and to derive an objective score for internal assessment.

: undergraduate medical examination is undergoing extensive re evaluation with new core educational objectives being defined. consequently, new exam systems have also been designed to test the objectives. objective structured practical examination (ospe) is one of them.objectives:to introduce ospe as a method of assessment of practical skills and learning and to determine student satisfaction regarding the ospe. furthermore, to explore the faculty perception of ospe as a learning and assessment tool.materials and methods: the first m.b.b.s students of 2011 12 batch of medical college, kolkata, were the subjects for the study. ospe was organized and conducted on identification of unknown abnormal constituents in urine. coefficient of reliability of questions administered was done by calculating cronbach's alpha. a questionnaire on various components of the ospe was administered to get the feedback.:16 students failed to achieve an average of 50% or above in the assessment. however, 49 students on an average achieved > 75%, 52 students achieved between 65% and 75%, and 29 students scored between 50% and 65%. cronbach's alpha of the questions administered showed to be having high internal consistency with a score of 0.80. ninety nine percent of students believed that ospe helps them to improve and 81% felt that this type of assessment fits in as both learning and evaluation tools. faculty feedback reflected that such assessment tested objectivity, measured practical skills better, and eliminated examiner bias to a greater extent.:ospe tests different desired components of competence better and eliminated examiner bias. student feedback reflects that such assessment helps them to improve as it is effective both as teaching and evaluation tools.

an ongoing outcomes follow - up project with a prospective inception cohort in western canada was started in 1996. as described previously, infants were identified at the time of complex cardiac surgery and were followed prospectively.11 in this study, we included all infants aged 6 weeks who were admitted to the pediatric intensive care unit after surgery for complex chd (defined as requiring cardiopulmonary bypass) at the stollery children s hospital between september 1996 and february 2009. demographic, preoperative, intraoperative, and postoperative variables that were previously agreed on were collected prospectively.11 chromosomal abnormalities were identified by routine karyotype and analysis for del22q11.2 in all infants and as clinically indicated up to age of follow - up. sepsis was defined as a positive blood culture that was treated for at least 5 days with intravenous antibiotics. potential skin contaminants in the blood culture (coagulase - negative staphylococci, aerococcus spp, micrococcus spp, corynebacterium spp, propionibacterium spp, viridians group streptococci, or bacillus spp)12 required 2 positive blood cultures or 1 positive blood culture associated with fever or hypothermia, abnormal white blood cell count, and increase in inotrope score on the day of the culture. the patients with potential skin contaminants in the blood culture were identified as those with a gram - positive organism grown from the culture. all of these patients had their charts reviewed retrospectively to confirm the above criteria; sepsis was confirmed in 41 of 57 (72%) and ruled out in the others. pragmatically, our definition of sepsis was meant to include children with clinical sepsis (ie, had an indication for the clinician to perform blood culture and to decide to treat with intravenous antibiotics for at least 5 days) with bacteremia and did not capture children that may have had clinical sepsis without proven bacteremia. we were able to review all required charts to confirm the criteria, and we cross - checked them with our hospital infection control nosocomial bacteremia database. long - term follow - up was performed, with parental or guardian consent, during the respective follow - up visits at the tertiary site of origin. the follow - up study and database were approved by the institutional health research ethics board. outcomes assessments were completed at a mean of 55 months (sd 6 ; median 54 months, interquartile range 51 to 59 months) of age. pediatric psychologists administered the wechsler preschool and primary scale of intelligence, revised (38 children tested prior to 2002) and third edition (358 children tested from 2002 onward), to obtain the full - scale intelligence quotient (fsiq), the performance intelligence quotient (piq), and the verbal intelligence quotient (viq) and administered the beery - buktenica developmental test of visual motor integration (vmi), fifth edition; the of the adaptive behavior assessment system, second edition (abas - ii) were obtained at the respective referral institutions.1316 the preschool version of abas - ii is a parent / caregiver - completed questionnaire to provide comprehensive, norm - referenced assessment of adaptive skills for children aged 0 to 5 years. the general adaptive composite (gac) score from the abas - ii is used to assess adaptive function in children. all scores have a normative population mean of 100 and sd of 15, and higher scores indicate better performance; a score < 70 is 2 sd below the mean and is expected in 2.27% of the normative population. continuous variables are presented as mean (sd), and categorical variables are presented as counts (percentages). to screen for variables associated with the outcome variables, we used univariate regression models and included the following a priori specified variables from the respective perioperative time periods: demographic (gestational age, birth weight, socioeconomic status,17 sex, chromosomal abnormality, and single ventricle anatomy), preoperative (5-minute apgar score, year of surgery), intraoperative (cardiopulmonary bypass time, aortic cross - clamp time, and deep hypothermic circulatory arrest used), postoperative (day 1 highest lactate, day 1 inotrope score,18 day 1 highest creatinine, day 1 highest base deficit, and the time for lactate to fall to 2 mmol / l), and all perioperative time periods (seizures, cardiopulmonary resuscitation, dialysis, sepsis, and extracorporeal membrane oxygenation anytime up to age 4 years). to screen for variables associated with neurocognitive outcomes, we used univariate regression models. multiple linear regression models consisted of variables found approaching significance at p0.10 in the univariate analysis and after screening for multicollinearity; they are presented as regression coefficients with 95% cis and 2-sided p values. the regression coefficient represents the average change in the outcome for each unit increase in the explanatory (predictor) variable. multiple cox proportional hazards regression analysis for mortality by 4.5 years included variables that were significant at p0.10 in the corresponding univariate analysis and after screening for multicollinearity, and they are presented as hazard ratios with 95% cis and 2-sided p values. low counts for some variables did not allow validation of the proportionality assumption in cox models. multicollinearity was assessed using pearson correlation for continuous variables and, when at least 1 of the variables was binary, using spearman correlation; pairs of variables with a correlation > 0.7 were declared collinear, and only 1 was entered into the multiple model according to clinical and statistical significance.19 using variance inflation factor as a regression diagnostic measure did not change the findings for collinearity.19 the statistical modeling is exploratory, investigating the effect of prespecified clinically and biologically important variables (sepsis and ecmo) while controlling for other potentially predictive confounding variables (those found significant on univariate analyses). we hypothesized that sepsis and ecmo would be associated with outcomes on multivariable analyses based on previous studies.19,20,21 statistical analyses were performed using sas version 9.3 (sas institute). the primary outcomes for this study were the intelligence quotient (fsiq, piq, viq) outcomes in survivors. outcomes assessments were completed at a mean of 55 months (sd 6 ; median 54 months, interquartile range 51 to 59 months) of age. pediatric psychologists administered the wechsler preschool and primary scale of intelligence, revised (38 children tested prior to 2002) and third edition (358 children tested from 2002 onward), to obtain the full - scale intelligence quotient (fsiq), the performance intelligence quotient (piq), and the verbal intelligence quotient (viq) and administered the beery - buktenica developmental test of visual motor integration (vmi), fifth edition; the of the adaptive behavior assessment system, second edition (abas - ii) were obtained at the respective referral institutions.1316 the preschool version of abas - ii is a parent / caregiver - completed questionnaire to provide comprehensive, norm - referenced assessment of adaptive skills for children aged 0 to 5 years. the general adaptive composite (gac) all scores have a normative population mean of 100 and sd of 15, and higher scores indicate better performance; a score < 70 is 2 sd below the mean and is expected in 2.27% of the normative population. continuous variables are presented as mean (sd), and categorical variables are presented as counts (percentages). to screen for variables associated with the outcome variables, we used univariate regression models and included the following a priori specified variables from the respective perioperative time periods: demographic (gestational age, birth weight, socioeconomic status,17 sex, chromosomal abnormality, and single ventricle anatomy), preoperative (5-minute apgar score, year of surgery), intraoperative (cardiopulmonary bypass time, aortic cross - clamp time, and deep hypothermic circulatory arrest used), postoperative (day 1 highest lactate, day 1 inotrope score,18 day 1 highest creatinine, day 1 highest base deficit, and the time for lactate to fall to 2 mmol / l), and all perioperative time periods (seizures, cardiopulmonary resuscitation, dialysis, sepsis, and extracorporeal membrane oxygenation anytime up to age 4 years). to screen for variables associated with neurocognitive outcomes, we used univariate regression models. multiple linear regression models consisted of variables found approaching significance at p0.10 in the univariate analysis and after screening for multicollinearity; they are presented as regression coefficients with 95% cis and 2-sided p values. the regression coefficient represents the average change in the outcome for each unit increase in the explanatory (predictor) variable. multiple cox proportional hazards regression analysis for mortality by 4.5 years included variables that were significant at p0.10 in the corresponding univariate analysis and after screening for multicollinearity, and they are presented as hazard ratios with 95% cis and 2-sided p values. low counts for some variables did not allow validation of the proportionality assumption in cox models. multicollinearity was assessed using pearson correlation for continuous variables and, when at least 1 of the variables was binary, using spearman correlation; pairs of variables with a correlation > 0.7 were declared collinear, and only 1 was entered into the multiple model according to clinical and statistical significance.19 using variance inflation factor as a regression diagnostic measure did not change the findings for collinearity.19 the statistical modeling is exploratory, investigating the effect of prespecified clinically and biologically important variables (sepsis and ecmo) while controlling for other potentially predictive confounding variables (those found significant on univariate analyses). we hypothesized that sepsis and ecmo would be associated with outcomes on multivariable analyses based on previous studies.19,20,21 statistical analyses were performed using sas version 9.3 (sas institute). the primary outcomes for this study were the intelligence quotient (fsiq, piq, viq) outcomes in survivors. there were 502 consecutive infants enrolled in the inception cohort at the time of cardiac surgery at age 6 weeks. demographics included 315 (63%) male patients, 160 (32%) with single ventricle anatomy, 49 (10%) having ecmo at any time up to age 4.5 years, and 45 (9%) with chromosomal abnormality. perioperative sepsis occurred in 97 patients (19%) overall and in 76 of 396 survivors (19%) with 4.5-year follow - up. by 4.5 years of age , there were 91 deaths (18%), and 396 of 411 survivors (96%) had neurocognitive follow - up completed (figure). regarding mortality, 31 (6.2%) and 86 (17%) patients had died at 30 days and 2 years, respectively. there was no evidence of collinearity between sepsis and ecmo at any time (correlation 0.03, p = 0.48). the mean neurocognitive outcomes in survivors at 4.5 years of age included fsiq 90.6 (sd 18.6), viq 90.8 (sd18.6), piq 91.4 (sd 19.2), vmi 89.7 (sd 16.7), and gac 89.6 (sd 18.1). scores < 70 on both fsiq and gac occurred in 43 patients (10.9%); therefore, the neurocognitive outcomes shifted to the left of the normative population, with more than expected having scores > 2 sd below the mean. description of the cohort of 502 patients having cardiac surgery in early infancy ecmo indicates extracorporeal membrane oxygenation; fsiq, full - scale intelligence quotient; gac, general adaptive composite; iqr, interquartile range; piq, performance intelligence quotient; viq, verbal intelligence quotient; vmi, beery - buktenica developmental test of visual motor integration. perioperative sepsis occurred in 97 patients, 29 preoperatively, 75 postoperatively, and 7 both pre- and postoperatively. in the sepsis subgroup, 12 of 97 (12%) had ecmo and 11 of 97 (11%) had chromosomal abnormalities. of the 76 sepsis survivors, 5 (7%) had ecmo and 7 (9%) had chromosomal abnormality. flow diagram of the inception cohort and analyses for mortality and neurocognitive outcomes. on multiple cox proportional hazards regression analysis, sepsis was not and ecmo was associated with mortality by 4.5 years (hazard ratio 1.9 ; 95% ci 1.17 to 3.18 ; p = 0.01) (table 2). univariate and multiple cox proportional hazards regressions for death at 4.5 years in 502 patients having cardiac surgery in early infancy cpb indicates cardiopulmonary bypass; cpr, cardiopulmonary resuscitation; ecmo, extracorporeal membrane oxygenation; hr, hazard ratio. mean fsiq, piq, and viq scores at 4.5 years of age for the 76 sepsis survivors were 84.8 (sd 17.5), 84.8 (sd 17.6), and 85.8 (sd 17.7), respectively; these scores are statistically significantly lower than the respective scores in the rest of the cohort (92 , p = 0.002 ; 93 , p = 0.001 ; and 92 , p = 0.009). univariate and multivariable predictors of the fsiq, viq, and piq outcomes are given in tables3 and 4, respectively. chromosomal abnormality was associated with worse scores on all of these outcomes on multivariable analyses. ecmo anytime in the first 4 years was also strongly associated with worse scores on all of these outcomes on multivariable analyses; for fsiq, the regression coefficient was 13.6 (95% ci 21.3 to 5.9 ; p = 0.001). sepsis perioperatively was associated with piq and viq, with a trend toward significance for fsiq (p = 0.058) on multivariable analyses. the regression coefficient for sepsis was strongest for piq (5.31 ; 95% ci 9.84 to 0.78 ; p = 0.022). other variables associated with 2 iq outcomes on multivariable analyses included socioeconomic status, single ventricle anatomy, and sex. post hoc exploration for an interaction between sepsis and single ventricle anatomy found that interaction terms were not statistically significant. univariate linear regressions for full - scale intelligence quotient, performance intelligence quotient, and verbal intelligence quotient at 4.5 years of age in 396 survivors of cardiac surgery in early infancy ecmo indicates extracorporeal membrane oxygenation; fsiq, full - scale intelligence quotient; piq, performance intelligence quotient; viq, verbal intelligence quotient. multivariable linear regressions for full - scale intelligence quotient, performance intelligence quotient, and verbal intelligence quotient at 4.5 years of age in 396 survivors of cardiac surgery in early infancy r indicates the variability in the outcomes scores accounted for by the model. ecmo indicates extracorporeal membrane oxygenation; fsiq, full - scale intelligence quotient; piq, performance intelligence quotient; viq, verbal intelligence quotient. univariate and multivariable predictors of the secondary outcomes of vmi and gac are given in tables5 and 6. ecmo anytime in the first 4.5 years was also strongly associated with worse scores on multivariable analyses, with regression coefficients of 13.4 (95% ci 20.1 to 6.7 ; p<0.001) and 12.7 (95% ci 20.3 to 5.0 ; p = 0.001) for vmi and gac, respectively. other predictors on multivariable analysis for gac included birth weight and sex and for vmi included birth weight and single ventricle anatomy. univariate linear regressions for general adaptive composite of the adaptive behavioral assessment system and visual motor integration at 4 years of age in 396 survivors of cardiac surgery in early infancy ecmo indicates extracorporeal membrane oxygenation. multivariable linear regressions for general adaptive composite and visual motor integration at 4 years of age in 396 survivors of cardiac surgery in early infancy r indicates the variability in the outcomes scores accounted for by the model. post hoc, we explored whether excluding children who had ecmo at any time in the first 4.5 years would change the for the primary outcomes. we hypothesized that ecmo could mask other predictors that may be important in determining outcomes. there was no evidence of collinearity between sepsis and ecmo (correlation 0.03, p = 0.48). excluding children who had ecmo did not significantly change the for any of the primary outcomes (data not shown). sepsis remained a predictor of viq and piq, and the trend for fsiq remained (p = 0.079) on multivariable analyses. the only new association on multivariable analysis was between socioeconomic status and piq (regression coefficient 0.14 ; 95% ci 0.01 to 0.28 ; p = 0.039). there were 502 consecutive infants enrolled in the inception cohort at the time of cardiac surgery at age 6 weeks. demographics included 315 (63%) male patients, 160 (32%) with single ventricle anatomy, 49 (10%) having ecmo at any time up to age 4.5 years, and 45 (9%) with chromosomal abnormality. perioperative sepsis occurred in 97 patients (19%) overall and in 76 of 396 survivors (19%) with 4.5-year follow - up. by 4.5 years of age , there were 91 deaths (18%), and 396 of 411 survivors (96%) had neurocognitive follow - up completed (figure). regarding mortality, 31 (6.2%) and 86 (17%) patients had died at 30 days and 2 years, respectively. there was no evidence of collinearity between sepsis and ecmo at any time (correlation 0.03, p = 0.48). the mean neurocognitive outcomes in survivors at 4.5 years of age included fsiq 90.6 (sd 18.6), viq 90.8 (sd18.6), piq 91.4 (sd 19.2), vmi 89.7 (sd 16.7), and gac 89.6 (sd 18.1). scores < 70 on both fsiq and gac occurred in 43 patients (10.9%); therefore, the neurocognitive outcomes shifted to the left of the normative population, with more than expected having scores > 2 sd below the mean. description of the cohort of 502 patients having cardiac surgery in early infancy ecmo indicates extracorporeal membrane oxygenation; fsiq, full - scale intelligence quotient; gac, general adaptive composite; iqr, interquartile range; piq, performance intelligence quotient; viq, verbal intelligence quotient; vmi, beery - buktenica developmental test of visual motor integration. perioperative sepsis occurred in 97 patients, 29 preoperatively, 75 postoperatively, and 7 both pre- and postoperatively. in the sepsis subgroup, 12 of 97 (12%) had ecmo and 11 of 97 (11%) had chromosomal abnormalities. of the 76 sepsis survivors, 5 (7%) had ecmo and 7 (9%) had chromosomal abnormality. flow diagram of the inception cohort and analyses for mortality and neurocognitive outcomes. on multiple cox proportional hazards regression analysis, sepsis was not and ecmo was associated with mortality by 4.5 years (hazard ratio 1.9 ; 95% ci 1.17 to 3.18 ; p = 0.01) (table 2). univariate and multiple cox proportional hazards regressions for death at 4.5 years in 502 patients having cardiac surgery in early infancy cpb indicates cardiopulmonary bypass; cpr, cardiopulmonary resuscitation; ecmo, extracorporeal membrane oxygenation; hr, hazard ratio. mean fsiq, piq, and viq scores at 4.5 years of age for the 76 sepsis survivors were 84.8 (sd 17.5), 84.8 (sd 17.6), and 85.8 (sd 17.7), respectively; these scores are statistically significantly lower than the respective scores in the rest of the cohort (92 , p = 0.002 ; 93 , p = 0.001 ; and 92 , p = 0.009). univariate and multivariable predictors of the fsiq, viq, and piq outcomes are given in tables3 and 4, respectively. chromosomal abnormality was associated with worse scores on all of these outcomes on multivariable analyses. ecmo anytime in the first 4 years was also strongly associated with worse scores on all of these outcomes on multivariable analyses; for fsiq, the regression coefficient was 13.6 (95% ci 21.3 to 5.9 ; p = 0.001). sepsis perioperatively was associated with piq and viq, with a trend toward significance for fsiq (p = 0.058) on multivariable analyses. the regression coefficient for sepsis was strongest for piq (5.31 ; 95% ci 9.84 to 0.78 ; p = 0.022). other variables associated with 2 iq outcomes on multivariable analyses included socioeconomic status, single ventricle anatomy, and sex. post hoc exploration for an interaction between sepsis and single ventricle anatomy found that interaction terms were not statistically significant. univariate linear regressions for full - scale intelligence quotient, performance intelligence quotient, and verbal intelligence quotient at 4.5 years of age in 396 survivors of cardiac surgery in early infancy ecmo indicates extracorporeal membrane oxygenation; fsiq, full - scale intelligence quotient; piq, performance intelligence quotient; viq, verbal intelligence quotient. multivariable linear regressions for full - scale intelligence quotient, performance intelligence quotient, and verbal intelligence quotient at 4.5 years of age in 396 survivors of cardiac surgery in early infancy r indicates the variability in the outcomes scores accounted for by the model. ecmo indicates extracorporeal membrane oxygenation; fsiq, full - scale intelligence quotient; piq, performance intelligence quotient; viq, verbal intelligence quotient. univariate and multivariable predictors of the secondary outcomes of vmi and gac are given in tables5 and 6. ecmo anytime in the first 4.5 years was also strongly associated with worse scores on multivariable analyses, with regression coefficients of 13.4 (95% ci 20.1 to 6.7 ; p<0.001) and 12.7 (95% ci 20.3 to 5.0 ; p = 0.001) for vmi and gac, respectively. other predictors on multivariable analysis for gac included birth weight and sex and for vmi included birth weight and single ventricle anatomy. univariate linear regressions for general adaptive composite of the adaptive behavioral assessment system and visual motor integration at 4 years of age in 396 survivors of cardiac surgery in early infancy ecmo indicates extracorporeal membrane oxygenation. multivariable linear regressions for general adaptive composite and visual motor integration at 4 years of age in 396 survivors of cardiac surgery in early infancy r indicates the variability in the outcomes scores accounted for by the model. post hoc, we explored whether excluding children who had ecmo at any time in the first 4.5 years would change the for the primary outcomes. we hypothesized that ecmo could mask other predictors that may be important in determining outcomes. there was no evidence of collinearity between sepsis and ecmo (correlation 0.03, p = 0.48). excluding children who had ecmo did not significantly change the for any of the primary outcomes (data not shown). sepsis remained a predictor of viq and piq, and the trend for fsiq remained (p = 0.079) on multivariable analyses. the only new association on multivariable analysis was between socioeconomic status and piq (regression coefficient 0.14 ; 95% ci 0.01 to 0.28 ; p = 0.039). first, perioperative sepsis, defined as a positive blood culture not due to a contaminated specimen and treated with intravenous antibiotics for at least 5 days, was associated with adverse long - term viq and piq (and with a trend to adverse fsiq ; p = 0.058) outcomes on multivariable analyses. second, ecmo at any time in the first 4.5 years of life was associated with adverse outcomes on all fsiq, viq, piq, vmi, and gac scores on multivariable analyses. third, these associations on multivariable analyses were found after controlling for other prespecified contributors to adverse outcomes, including severity of illness (eg, lactate, inotrope score), demographics (eg, birth weight, sex, socioeconomic status, chromosomal abnormality, single ventricle anatomy), and other perioperative events (eg, deep hypothermic circulatory arrest, cardiopulmonary resuscitation, seizures). in fact, several of these variables were also associated with some of the adverse neurocognitive outcomes on multivariable analyses, including birth weight, socioeconomic status, single ventricle anatomy, and chromosomal abnormality. of particular importance, sepsis was the only consistently identified, potentially modifiable variable associated with the neurocognitive outcomes on multivariable analyses. the only other potentially modifiable variables associated with neurocognitive outcomes on multivariable analyses reflected early postoperative severity of illness: postoperative time for lactate to fall 2 mmol / l and postoperative day 1 inotrope score were associated with adverse viq and gac, respectively. the finding of the association between sepsis and adverse neurocognitive outcomes on multivariable analyses is consistent with the adult literature14 and strengthens the existing pediatric literature59 by using a large sample size in a prospective inception cohort (n = 396 survivors), a high rate of follow - up (96% of survivors followed to 4.5 years of age), a detailed neurocognitive assessment done with validated standardized testing,1316 and adjustment for many potentially confounding variables. that sepsis is a potentially modifiable predictor of adverse outcomes is supported by literature on the prevention of sepsis in the critical care environment.22,23 this is also important because sepsis is increasingly common in the pediatric intensive care unit.2426 the finding of the association between ecmo and adverse neurocognitive outcomes on multivariable analyses has been suspected before but, to our knowledge, has not been confirmed in a large cohort, adjusting for potential confounders.20,21 a recent review found that neurocognitive outcomes after ecmo in children has been examined in only a few small studies, often without detailed neurocognitive assessment and/or comparison to control groups with similar demographics and surgery.20 this study has limitations. first, it is an observational study, and we can not claim a cause - and - effect relationship between predictors and outcomes. second, our definition of sepsis requiring a positive blood culture may have missed many cases of nonbacteremic sepsis. in addition , we can not confirm that all of our patients had the systemic inflammatory response syndrome, part of the definition of sepsis, because we did not record temperature, respiratory rate, heart rate, or white blood cell count for all of the included children. third, although patients were enrolled and had variables recorded prospectively, the confirmation of blood culture was done retrospectively. fourth, the rates of perioperative blood culture positive sepsis were high.27 hospital - acquired infection rates in pediatric patients undergoing surgical correction of chd has been reported to be 16%, with bloodstream infection rates of 5% to 10%.27 fifth, a small number of the children at the beginning of the study were tested with an earlier edition of the wechsler preschool and primary scale of intelligence test for neurocognitive outcomes. data collection for this study extended for over 11 years, during which time the intelligence test was revised. the option of continuing with the outdated test was rejected based on the principle of using the up - to - date version. with no difference between the means (sds) of the 2 editions within the normative population and within a previously published cohort from our group , we proceeded to include all children in the cohort.28 sixth, sepsis was not associated with ecmo; it is possible that the severity of the underlying chd and residual cardiac lesions, postoperative complications including cardiac arrest and low cardiac output, and our definition of sepsis not capturing many children with clinical sepsis (ie, without bacteremia) may have confounded any impact of sepsis on need for ecmo. finally, because this study explored the predictors of outcome in a large cohort of children, all of whom had surgery in early infancy for chd, we can not comment on outcomes or predictors of outcomes in children with chd who did not have surgery in early infancy. nevertheless, the multivariate analyses done controlled for prespecified potential predictors of outcome identified in previous studies,11 and the criteria used to confirm blood culture were prespecified and objective, modified from the centers for disease control and prevention definitions.12 furthermore, we included a group of infants at high risk for sepsis, having complex surgery for chd at age 6 weeks, requiring many days of pre- and postoperative intensive care, and often having central venous lines and intubation for much of that time.27 importantly, the high rate of sepsis suggests that known systems and interventions to prevent nosocomial infections have the potential to improve long - term neurocognitive outcomes in these children.22,23 the association of sepsis with adverse neurocognitive outcomes may be an underestimate, given that many cases of nonbacteremic sepsis likely occurred. our statistical approach was to use multivariable analyses to explore for predictors of outcome while adjusting for clinically (sepsis and ecmo) and statistically (on univariate analysis) important potentially confounding variables. in a large prospective inception cohort of patients having complex cardiac surgery early in infancy , we found that perioperative sepsis was associated with adverse neurocognitive outcomes on multivariable analyses. having received ecmo at any time in the first 4 years of life was also found to be strongly associated with adverse neurocognitive outcomes on multivariable analyses. nosocomial perioperative sepsis is a potentially modifiable variable, and these data highlight the importance of infection control practices in intensive care. the complex pediatric therapies follow - up program has been supported over the years by alberta health, the stollery children s hospital, and the glenrose rehabilitation hospital. the women and children s health research institute has funded acute care data collection starting in 2014. these funding agencies had no role in the design and conduct of the study; collection, management, analysis or interpretation of the data; preparation, review, or approval of the manuscript; or the decision to submit the manuscript for publication.

we aimed to determine whether sepsis is associated with neurocognitive outcomes 4.5 years after congenital heart disease surgery in early infancy.methods and a secondary analysis from a prospective inception cohort included all children having congenital heart disease surgery done at 6 weeks of age with cardiopulmonary bypass at the western canadian referral center from 1996 to 2009. follow - up at the referral center determined the primary outcomes at 4.5 years with full - scale, performance, and verbal intelligence quotients on the wechsler preschool and primary scale of intelligence. perioperative variables were collected prospectively, and confirmation of blood culture positive sepsis was done retrospectively. multiple linear regression models for neurocognitive outcomes and multiple cox proportional hazards regression for mortality were determined. sepsis occurred in 97 of 502 patients (19%) overall and in 76 of 396 survivors (19%) with 4.5-year follow - up. by 4.5 years , there were 91 (18%) deaths, and 396 of 411 survivors (96%) had follow - up completed. extracorporeal membrane oxygenation was associated with worse scores on all neurocognitive outcomes on multivariable regression; the association between extracorporeal membrane oxygenation and full - scale intelligence quotient had a regression coefficient of 13.6 (95% ci 21.3 to 5.9 ; p = 0.001). sepsis perioperatively was associated with performance and verbal intelligence quotients, with a trend for full - scale intelligence quotient (p = 0.058) on multivariable regression. the regression coefficient for sepsis was strongest for performance intelligence quotient (5.31 ; 95% ci 9.84 to 0.78 ; p = 0.022). sepsis was not but extracorporeal membrane oxygenation was associated with mortality by 4.5 years.perioperative sepsis and extracorporeal membrane oxygenation were associated with adverse neurocognitive outcomes on multivariable regression. quality improvement to prevent sepsis has the potential to improve long - term neurocognitive outcomes in infants after surgery for congenital heart disease.

hypertension is an important worldwide public - health challenge because it is one of the most common chronic conditions. the prevalence of hypertension in sub - saharan africa is between 12.5 and 26.9%. in 2005 the world health organization stepwise approach to surveillance of noncommunicable diseases risk factors established prevalence of hypertension in cte d'ivoire to 21.7%. hypertension is a major risk factor for cardiovascular (cv) disease. it remains an important cause of coronary heart disease, cerebrovascular disease, peripheral artery disease, and heart failure. age is the most powerful risk factor for hypertension, death, and cardiovascular death. the worldwide increase in the elderly population (age 65 years) is associated with concurrent increases in prevalence of systemic hypertension and morbidity and mortality from vascular complications of hypertensive disease. recently, numerous large clinical trials have provided evidence of the benefits of reducing bp in the elderly. meta - analysis of clinical trials showed that treatment of hypertension in older adults is as beneficial as that in younger adults. it is well established now that the treatment of hypertension in elderly patient was associated with a reduction in the rate of fatal or nonfatal stroke, a reduction in the rate of death from stroke, a reduction in the rate of death from any cause, a reduction in the rate of death from cardiovascular causes, and a reduction in the rate of heart failure. it was in this context that we undertook the present study at institute of cardiology of abidjan, the single university hospital managing cardiovascular diseases in cte d'ivoire. this study aims to describe characteristics, risk factors, treatment and control of blood pressure of elderly hypertensive patients. we undertook a retrospective descriptive study involving patients seen at the outpatient clinics of the institute of cardiology of abidjan. the study population was hypertensive elders (aged at least 65 years) with a regular followup at the institute of cardiology of abidjan within one year. this series includes patients who had been on an initial treatment upon referral to our center. we used the definition and classification hypertension standards of the european society of hypertension (esh) and the european society of cardiology (esc). we have separated the systolic - diastolic hypertension from isolated systolic hypertension and from isolated diastolic hypertension. the data collected were age, sex, blood pressure at initial presentation and during the followup (one month, 2 months, 3 months, 6 months, 1 year or last presentation), the coexistence of other cardiovascular risk factors, the impact of hypertension, and the treatment modalities. the following additional cv risk factors were document when present: current smoking, dyslipidaemia, diabetes, and obesity. dyslipidaemia was defined according to our biochemistry laboratory standard when the level of total cholesterol was > 2 g / dl, ldl - cholesterol > 1.4 g / dl, or hdl - cholesterol < 0.4 g / l. new diabetic patients detected at institute of cardiology of abidjan were diagnosed based on the standard value in our laboratory, fasting plasma glucose > 1.26 g / dl on repeated measurement. the overall management of diabetes was coordinated by the individual patient's physician and not by the cardiology staff of institute of cardiology of abidjan. echocardiogram was frequently performed, and the were included in the data collection. finally, when other tests were seldom performed (e.g., albuminuria, microalbuminuria, the fundscopy, and 24-hour ambulatory blood pressure), they were not used for analysis. the esh and esc categorization of total risk as low, moderate, high, and very high added risk has the merit of simplicity and was, therefore, chosen for risk stratification.. blood pressure control was defined as a treated blood pressure < 140 mmhg systolic and < 90 mmhg diastolic and was ascertained by direct measurement of blood pressure. during the study period, 2575 hypertensive black africans patients have had a regular follow - up of at least one year. among these patients, the mean age was 73.1 5.3 years (range 6598 years), 59% were female. at first presentation blood pressure it was mostly systolic - diastolic hypertension (51.8%) and isolated systolic hypertension (38.5%). mean blood pressure was 169.4 28.4 mmhg for systolic, 95.3 15.7 mmhg for diastolic, and 74.1 22.8 mmhg for pulse pressure. blood pressure of men was not significantly different from that of women (table 2). overall, patients were diagnosed with hypertension for an average of 4 6.7 years (range 147) at the time of their initial assessment in our clinics. the mean follow - up duration in ica was 3 1.8 years (range 1 to 18 years). isolated cardiac complications were the most frequent (37.9%). the cardiovascular risk factors other than hypertension and the of target organ damage are reported in table 1. the recorded anomalies were a left ventricular diastolic dysfunction (39.2%), hypertensive cardiomyopathy (32.1%) and minor lesions (slight valve regurgitation, valvular sclerosis, valvular calcification) (19.6%). application of the cardiovascular risk stratification according to the european guideline for the management of arterial hypertension identified a very high added risk in 82.1%, a high added risk in 4.8%, a moderate add risk in 7.5% and a low add risk in 5.7%. in addition to lifestyle changes, 93.5% of patients received antihypertensive drugs (table 2). of those on treatment for high blood pressure, the most common agents used were diuretics in 63.5%, followed by blockers of the renin - angiotensin system (ras) in 61.3% (either angiotensin converting enzyme inhibitors or angiotensin receptor blockers), calcium antagonists (31.6%), -blockers (19%), and centrally acting sympatholytics (4.5%). antihypertensive drugs were used in monotherapy or combination (table 3). as a monotherapy, the most prescribed drug class was calcium antagonists (36.3%), followed by ras blocker (32%). polytherapy (more than 2 antihypertensive drugs including fixed - dose combination drugs) was used for 67% of patients on treatment for hypertension. the most common combination of drugs among those taking 2 agents was ras blockers plus diuretics (69.8%). diabetic patients as well as those with kidney failure have received significantly more ras - blockers for the treatment of hypertension than other patients (p = 0.03 for diabetic and p = 0.04 for renal failure). between the first and the last visit, blood pressure decreased significantly (table 3). it was for systolic blood pressure by 15.9 mmhg, diastolic blood pressure by 8.2 mmhg, and pulse pressure by 7.6 mmhg. the sex (p = 0.88), age (p = 0.48), duration of treatment of hypertension (p = 0.13), the number of consultation (p = 0.13), and the duration of hypertension (p = 0.27) did not influence the control of blood pressure. hypertension was best controlled with multiple drugs therapy (1.7 drugs for controlled patients as compared to 1.4 for to uncontrolled patients, p < 0.001). pulse pressure was significantly lowered in females (p = 0.03) (table 4). this study found that a significant proportion of patients (33%) who visit the ica are elderly. in the elderly a significant part of hypertension is represented by systolic hypertension. increased arterial stiffness may increase cardiovascular morbidity and mortality because of an elevation of systolic blood pressure (sbp), which raises left ventricular afterload, and because of a decrease in diastolic blood pressure (dbp), which alters coronary perfusion. elevated pp is a powerful independent predictor of cardiovascular end points in the elderly. according to skurnick et al., the pp levels of women were lower than those of men in early adulthood and higher in older ages. but in our study, although the difference was not statistically significant, the pp of men was higher than women's pp. moreover, under hypertension treatment, the pp of women had significantly regressed compared to men's. as has been previously described, it is suggested that black africans present more severe forms of arterial hypertension and a greater risk of target organ damage. overall, uncontrolled blood pressure remains the main factor for target organ damage more frequently in sub - saharan africa compared to western countries. the main benefits of antihypertensive treatment are blood pressure lowering per se, largely independent of the drugs employed. diuretics, -blockers, calcium antagonists, and ras blockers can adequately lower blood pressure, significantly improving cardiovascular outcome. several properties of the thiazide - type diuretics have led to them being recommended as first - line therapy in older adults with uncomplicated stage 1 hypertension. at low doses (< 25 mg / day of hydrochlorothiazide or equivalent), these agents have been demonstrated in randomized controlled trials to reduce mortality, stroke, and cardiovascular events in the older hypertensive population. there is good synergy with agents of different classes (ras - blockers and calcium antagonists) and most importantly in the elderly; these drugs preferentially lower sbp relative to dbp. in our study, diuretics have been widely prescribed. ras - blockers were also widely prescribed because of comorbidities such as diabetes, left ventricle hypertrophy or kidney failure, situations that required a preferential indication of ras - blockers. furthermore, hyvet recommended the addition of a ras - blocker in the event of insufficient control of bp. the ras - blocker diuretic combination was by far the most used in our study. in monotherapy, significant reductions in stroke risk in older hypertensive patients were demonstrated in the systolic hypertension europe and china trials. furthermore, from patients with very high cardiac risk enrolled in accomplish trial demonstrated the superiority of an acei - calcium antagonists (amlodipine) combination over an ace - thiazide combination with regard to a decrease in cardiovascular events despite comparable bp - lowering effects. in most cases, if blood pressure is more than 20/10 mmhg above the target bp, treatment should be initiated with two antihypertensive drugs. in our study, hypertension was best controlled with multiple drugs therapy. one should certainly not hesitate to use more than one antihypertensive drug even in elderly patients if the target blood pressure is not reached. control rate of hypertension (42.6%) was acceptable in our sub - saharan african context. there is no information on treatment tolerance, particularly on orthostatic hypotension occurrence in the elderly often polymedicated patients. also in these elderly patients with high cardiovascular risk, it would be interesting to describe cardiovascular events occurred during the year - long or more followup. furthermore, the control rate of hypertension obtained does not reflect the reality of the management of hypertension in cte d'ivoire. patients who were regularly monitored for at least one year showed probably best treatment adherence. despite the classical reduced life expectancy in sub - saharan african population because of various illnesses, numerous elderly people exist. the control rate of hypertension was significant, mostly at the cost of combination therapy.

. since the treatment of hypertension is beneficial for the elderly, we have undertaken this study that aims to evaluate the management of hypertension in elderly patient in cte d'ivoire. methods. a retrospective study was conducted among 854 hypertensive elderly patients of abidjan cardiology institute who were followed for a minimum of one year, between january 2000 and december 2009. . the patients mean age was 73.1 5.3 years, and 59% were women. at the first presentation, it was mostly systolic - diastolic hypertension (51.8%) and isolated systolic hypertension (38.5%). mean blood pressure was 169.4 28.4 mmhg for systolic, 95.3 15.7 mmhg for diastolic, and 74.1 22.8 mmhg for pulse pressure. pulse pressure was 60 mmhg in 80.4%. according to the european guidelines stratification of the cardiovascular risk - excess attributable to high blood pressure , 82.1% of the sample had a very high added risk. the pharmacological therapy was prescribed in 93.5%. more than 66% of patients were receiving 2 antihypertensive drugs including fixed - dose combination drugs. the most common agents used were diuretics (63.5%) followed by angiotensin - converting enzyme inhibitors or angiotensin receptor blockers in 61.3%. the most common agents used for monotherapy were calcium antagonists. when 2 drugs were used, diuretics and angiotensin - converting enzyme inhibitors or angiotensin receptor blockers were the most common. blood pressure control was achieved in 42.6%. . the control of elderly hypertension can be effective in sub - saharan africa. he required at least two antihypertensive drugs to meet the recommended blood pressure target.

in the case of older adults over 65 years of age, physiological changes caused by aging lead to much greater vulnerability with regard to safety issues compared to younger age groups1. due to physiological changes ing from aging, elders face a 10 times greater risk of falls than those in other age groups6. falls can happen in all age groups, but they especially occur in 1/3 of elders aged 65 or above, and about 50% of elders who have experienced falls experience recurrences2, 3. in more severe cases, elders lose independence in daily living because of aftereffects and declines in physical body functions4. it has been reported that the fear of such experiences influences daily living activities and mobility and that 4070% of elders face limitation in daily living due to this fear5. safely participating in meaningful daily living activities requires high - level, appropriate visual processing. although the visual perceptual skills of elders have a large impact on their daily living activities, there are still inadequacies in medical prevention systems and organized healthcare systems for elders, such as the early diagnosis and treatment of visual perceptual disorder6. position balance ability and most behaviors that occur in daily living are associated with visual perception7, and impairments in cognitive functioning affect the balance maintenance ability and physical body integration of elders, acting as a risk factor for falls8. balance is an essential element of functional activity, and balance ability is regarded as a critical factor involved in elders daily living9. therefore, this study selected a group of older adults at risk for falls and another of those not at risk for falls and evaluated their visual perceptual and cognitive functioning to check for a mild visual perceptual disorder unnoticed by them or their guardians. next, the study aimed to compare the elders balance maintenance ability, cognitive functioning, fall efficacy, and visual perceptual functioning, and to examine how these were correlated. this study was conducted from september 1, 2014, to march 30, 2015. in order to carry out the research using survey contents and assessment report measurements, all details of this study s procedures were submitted to the science research council of inje university, which approved the study protocol. this research was carried out on 116 elders aged 65 or above who use d seniors welfare center and y senior citizen center in busan. subjects were divided into a group of 64 elders at risk for falls and a group of 52 elders not at risk for falls using balance maintenance ability evaluation tools the berg balance scale and the one - legged stance test as well as a measurement tool on their knowledge of falls and whether or not they had experienced falls more than once over the past year. for the group at risk for falls, elders who could act in accordance with instructions, stand on their own, and walk for more than 10 m without outside help were selected from those capable of independent daily living10. before beginning the research, its overall content and expected effects were explained carefully to the elders, and only those who voluntarily consented participated. frequency analysis was performed using descriptive statistics for the general characteristics of the two subject groups, and t - tests were applied to analyze differences in balance maintenance ability, cognitive ability, fall efficacy, and visual perceptual functioning. in addition, pearson correlation analysis was applied to examine the relationships among balance maintenance ability, cognitive ability, fall efficacy, and visual perceptual functioning. to classify elders based on whether or not they were at risk for falls, subjects scoring less than 22 out of 30 on the falls knowledge measurement tool, 45 out of 56 on the berg balance scale, and 5 seconds on the one - legged stance test, were classified as being at risk for falls. the are in table 1table 1.knowledge of falls, berg balance scale, one - legged stance testcategorieselderly at risk of falls(n=64)elderly (n=52)m sdm sdknowledge of fall18.8 2.6 * 22.8 1.6*berg balance scale39.7 2.3 * 42.9 4.3*one - legged stance test: open(s) 2.0 0.9 * 5.3 2.1*one - legged stance test: off(s) 0.8 0.8 * 2.8 0.9*values are mean sd, * significant difference (p<0.05). values are mean sd, * significant difference (p<0.05) the cognitive functioning scores of the two groups as measured by the mmse - kc and moca - k are in table 2table 2.mmse-kc, moca - k scorecategoriesitem elderly at risk of falls (n=64)elderly (n=52)m sdm sdmmse - kctime orientation 3.5 1.53.7 0.8place orientation3.2 0.7 * 4.0 1.1*registration (three words)2.8 0.42.9 0.1attention and calculation (sam cheon ri gang san)1.2 1.3 * 3.0 1.1*recall (three words)0.7 0.9 * 1.4 1.2*naming1.3 0.8 * 2.3 0.7*three - stage command1.8 1.02.3 0.9copying interlocking pentagons0.6 0.40.5 0.5abstract thinking1.5 0.51.5 0.4total score17.2 5.3 * 22.1 3.8*moca - kvisuospatial / executive1.9 0.8 * 2.8 0.7*naming2.5 0.62.2 0.8attention2.4 1.13.7 1.1language2.2 0.72.3 0.5delayed recall1.7 4.319.3 2.3values are mean sd, * significant difference (p<0.05), and the fall efficacy scores of the two groups measured by the fes - k are shown in table 3table 3.fes-k scorem sdelderly at risk of falls (n=64)27.5 7.2*elderly (n=52)39.6 3.8*values are mean sd, * significant difference (p<0.05). finally, the visual perceptual function scores of the two groups are in table 4table 4.mvpt-3 scoreelderly at risk of falls (n=64)elderly (n=52)m sdm sdvisual short term memory 12.9 0.9 * 4.9 1.1*visual closure 14.0 1.0 * 6.5 2.1*spatial orientation1.4 0.8 * 3.0 0.7*figure ground1.1 0.7 * 2.2 0.9*visual closure 21.6 0.9 * 2.4 0.8*visual short term memory 21.3 0.81.5 1.1raw score26.5 2.7 * 34.8 5.4*standard score55.0 0.0 * 62.3 6.7*values are mean sd, * significant difference (p<0.05). values are mean sd, * significant difference (p<0.05) values are mean sd, * significant difference (p<0.05) values are mean sd, * significant difference (p<0.05) furthermore, the correlations among berg balance scale, one - legged test, mmse - kc, moca - k, fes - k, and mvpt-3 scores are shown in table 5table 5.correlations among balance maintenance ability, cognitive function, falls efficacy, and visual perceptual skillbbsolst (open)olst (close)mmse -kcmoca -kfes -kmvpt -3bbsolst (open)0.432**olst (close)0.205 * 0.588**mmse - k0.207 * 0.459**0.512**moca - k0.366**0.522**0.568**0.762**fes - k0.298**0.575**0.585**0.549**0.632**mvpt-30.349**0.624**0.485**0.442**0.518**0.506**values are mean sd, * significant difference (p<0.05), * * significant difference (p<0.01). bbs: berg balance scale; olst: one - legged stance test; mmse - kc: mmse in the korea version of the cered assessment packet; moca - k: korean version of the montreal cognitive assessment; fes - k: korea falls efficacy scale; mvpt: motor - free visual perception test. values are mean sd, * significant difference (p<0.05), * * significant difference (p<0.01). bbs: berg balance scale; olst: one - legged stance test; mmse - kc: mmse in the korea version of the cered assessment packet; moca - k: korean version of the montreal cognitive assessment; fes - k: falls in older adults are closely related to balance maintenance ability, cognitive functioning, and visual perceptual functioning. however, there are not enough studies examining the visual perception of elders or examining the correlations between falls or balance and visual perception. on the berg balance scale, both groups of older adults at risk for falls and those not at risk for falls had low scores (below 45), and both groups also scored low with 5 seconds on the one - legged stance test; both of these indicate that they were at risk for falls. park s research compared score differences between two groups of stroke patients, one with and one without fear of falls. there were significant differences between the two groups in the berg balance test and the one - legged stance test, but there was no significant difference in the fall efficacy criterion. the of this paper support those of the present study regarding the balance maintenance ability test carried out to classify older adults regarding their fall risk but do not agree with the of the present study regarding the falls efficacy criterion11. in this research, both groups showed a significant correlation of r=0.349 between their scores on the berg balance scale and the mvpt-3 test. there were significant correlations between the scores on the one - legged stance test with eyes open and the mvpt-3 test (r=0.624) and between the scores on the one - legged stance test with eyes closed and the mvpt-3 test (r=0.485). measuring the balance maintenance ability of elders helps to evaluate their ability to lead a safe life in their given environment, and physical stability and visual perceptual skills are important for this12. this research used the mvpt-3 test to evaluate the elders visual perceptual functioning. according to the , all subjects at risk for falls demonstrated very low - level visual perceptual functioning with an average raw score of m=26.53 (sd=2.7) and standard score of m=55.00 (sd=0.0). although subjects not at risk for falls had higher scores than those at risk for falls, these scores also showed their weak functioning. this indicates that community elders not at risk for falls also had a decline in visual perception due to normal aging processes. according to kim s research that analyzed mvpt raw scores to look into the overall visual perception conditions of community elders, elders in their 50s and 80s were all within the normal range, but elders in their 60s and 70s had lower scores than the normal range13. this means that healthy elders who have never received hospital treatments for visual perceptual disorder may have a visual perceptual disorder or decline in visual perceptual skills due to aging that they or their guardians have not noticed. the mvpt-3 raw scores of this study were low for both older adults at risk for falls and those not at risk for falls; this supports the present study, providing further evidence that even healthy elders may have a visual perceptual disorder or decline in visual perceptual ability that they or their guardians may not have noticed, as in previous research. cognitive functioning in this research was measured using the mmse - kc and the moca - k, and subjects had low scores in both tests, indicating severe cognitive disorder and mild cognitive disorder, respectively. this shows that there is a severe decline in the community elders cognitive functioning, which can also influence falls. the mmse - kc and mvpt-3 test scores had a significant correlation at r=0.442, and the moca - k and mvpt-3 test scores had a significant correlation at r=0.518. visual perception can be defined as an entire process of accepting and recognizing visual simulation14, and with a visual perception disorder, it is difficult to perceive objects and identify the mutual relation between objects in a space. moreover, the average fall efficacy scores (m=25.57, sd=7.2) of subjects at risk for falls were relatively low while of those not at risk for falls had higher average scores (m=39.69, sd=3.8). these support those of resnick, luisi, and vogel s research, which was conducted on elders residing in facilities and demonstrated that fall efficacy of subjects who experienced falls was significantly lower than in those who did not experience falls, showing that older adults at risk for falls have a great fear of falls9. such fear of falls causes a lack of confidence, decreases physical activities, and reduces the independent daily living of elders10. for the korean fall efficacy test, this research demonstrated significant correlations between balance maintenance ability, cognitive functioning, and visual perceptual function of elders. the falls efficacy test can be used clinically as a tool to predict elders fall risk in advance, thereby preventing falls. in addition, visual perceptual functions training should be reviewed to check whether it can improve visual perceptual function, balance maintenance ability, and cognitive functioning. the limitations of this research are that it recruited study subjects of high age groups from a local community senior citizen center, and it is difficult to generalize the to elders living in an active community.

this research aims to identify the relationships among visual perceptual skills, cognitive functioning, and fall efficacy of older adults based on whether they are at risk for falls. subjects included 116 older adults over 65 years of age who use d seniors welfare center and y senior citizen center in busan metropolitan city. all research subjects were classified based on balance maintenance ability evaluation and whether or not they had experienced falls more than once. those with scores below the cut - off standard were selected as a group of older adults at risk for falls. an mvpt-3 test was used to assess visual perceptual skill, mmse - kc, and moca - k tests to assess cognitive function, and the fes - k falls efficacy test to classify subjects as either at risk for falls or not. after comparing scores for visual perceptual skills, cognitive functioning, and fall efficacy, subjects at risk for falls showed significantly lower scores than did those not at risk. the study found that there are significant differences in balance ability, visual perceptual skill, cognitive functioning, and fall efficacy between older adults at risk for falls and those not at risk.