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the key role of the rho family gtpases rac , rho , and cdc42 in regulating the actin cytoskeleton is well established ( hall , a. 1998 . science . 279:509514 ) . increasing evidence suggests that the rho gtpases and their upstream positive regulators , guanine nucleotide exchange factors ( gefs ) , also play important roles in the control of growth cone guidance in the developing nervous system ( luo , l. 2000 . nat . rev . neurosci . 1:173180 ; dickson , b.j . 2001 . curr . opin . neurobiol . 11:103110 ) . here , we present the identification and molecular characterization of a novel dbl family rho gef , gef64c , that promotes axon attraction to the central nervous system midline in the embryonic drosophila nervous system . in sensitized genetic backgrounds , loss of gef64c function causes a phenotype where too few axons cross the midline . in contrast , ectopic expression of gef64c throughout the nervous system results in a phenotype in which far too many axons cross the midline , a phenotype reminiscent of loss of function mutations in the roundabout ( robo ) repulsive guidance receptor . genetic analysis indicates that gef64c expression can in fact overcome robo repulsion . surprisingly , evidence from genetic , biochemical , and cell culture experiments suggests that the promotion of axon attraction by gef64c is dependent on the activation of rho , but not rac or cdc42 .	during development , neuronal growth cones interpret a balance of attractive and repulsive cues present in the extracellular environment to find their correct targets . many phylogenetically conserved ligands and receptors that control axon guidance decisions have been discovered ( tessier - lavigne and goodman , 1996 ) . for example , in the embryonic drosophila central nervous system ( cns ) , * midline glia cells secrete netrin and slit ; netrin attracts axons across the midline , whereas slit repels axons , preventing them from crossing more than once ( harris et al . , 1996 ; netrin attraction is mediated by deleted in colo - rectal carcinoma ( dcc ) receptors and slit repulsion is mediated by roundabout ( robo ) receptors ( keino - masu et al . , 1996 ; kolodziej et al . , 1996 ; kidd et al . , chimeric receptors , generated by exchanging the cytoplasmic domains of the attractive netrin receptor dcc and the repulsive slit receptor robo , have shown that the sign of the growth cone response is encoded in the cytoplasmic domains of these receptors ( bashaw and goodman , 1999 ) . to identify signaling molecules involved in controlling axon guidance decisions , we have used chimeric receptor overexpression phenotypes to perform a sensitized genetic screen . overexpression of the robo - dcc chimeric receptor ( robo 's extracellular domain fused to dcc 's cytoplasmic domain ) leads to dose - dependent cns axon guidance defects in which axons abnormally cross the cns midline , and also results in reduced viability . we screened the ep collection ( a collection of p - element inserts that allow gal4-dependent misexpression of flanking genes [ rorth et al . , 1998 ] ) for genes that , when overexpressed pan - neurally in combination with robo - dcc , would enhance the viability defects of the chimera . such genes could play a role in dcc - mediated attractive axon guidance , or alternatively could function in parallel attractive - signaling pathways . here expression of ep3035 dramatically enhances the axon guidance defects of the robo - dcc chimera , leading to a significant increase in ectopic midline crossing ( unpublished data ) . molecular characterization of the genomic region adjacent to ep3035 revealed a large transcription unit that encodes a novel member of the dbl family of guanine nucleotide exchange factors ( gefs ) ( cerione and zheng , 1996 ) specific for the rho family of small gtpases ( fig . in addition to the canonical dbl and pleckstrin homology ( ph ) domains , gef64c also contains several proline - rich motifs , including a sequence similar to the enabled evh1 domain binding site ( lplpp ) ( niebuhr et al . , 1 ) . rna in situ analysis on ep3035/elavgal4 embryos confirms that ep3035 drives overexpression of the gef64c transcript . in addition , the genetic enhancement of robo - dcc by ep3035 can be phenocopied by expressing a uas gef64c transgene , confirming that the enhancement is due to gef64c expression ( unpublished data ) . protein expression analysis in wild - type embryos , using an mab to gef64c , reveals broad , low level expression of this gef , with some enrichment in the cns ( fig . the specificity of the gef64c mab is demonstrated by comparing embryos expressing full - length uasgef64c under control of elavgal4 , with those expressing a cooh - terminal truncation , uasgef64cc , which removes the mab epitope ( fig . robust cns expression can be seen in animals with the wild - type transgene , while only the low - levels characteristic of wild - type expression can be seen in animals with the truncated transgene ( fig . 1 , e and f ) . molecular characterization and expression of gef64c . the location of the ep insert , the sequenced mutant alleles , and the region used for antibody generation are indicated . colored regions of the coding sequence are as indicated in b. ( b ) schematic diagram of the gef64c and gef64cc proteins . the ph domain was identified by the smart sequence analysis program , but had a very low significance score ( 10 ) . since all known dbl domain proteins have ph domains flanking the dbl , we have included the ph domain with a small question mark next to it . sequences of identified domains are underlined in the color corresponding to each domain as indicated in b. ( d f ) stage 1516 embryos stained with anti - gef64c antibody . these sequence data are available from embl / genbank / ddbj under accession no . ay064174 . since gef64c was identified in a gain of function screen , we wanted to assess the consequence of loss of gef64c function on midline axon guidance . we generated point mutations in the gef and sequenced three independent alleles ( see materials and methods ) . two of these alleles , gef64c and gef64c , result in premature stop codons , while a third generates a missense mutation at the cooh terminus of the protein ( fig . embryos carrying mutations in gef64c were examined with an antibody that labels all cns axons ( mab bp102 ) . no major defects were discovered in the gef64c mutants : the longitudinal connectives and commissural axon bundles were comparable to those seen in wild - type animals ( fig . rna interference using a fragment of gef64c double - stranded rna also failed to reveal strong axon guidance defects , arguing against maternal contribution as an explanation for the absence of a mutant phenotype ( unpublished data ) . genetic redundancy could explain the modest consequences of removing gef64c ; indeed , examination of the drosophila genome reveals that there are 22 gefs specific for rho family gtpases , a number of which appear to be expressed in the embryonic cns ( unpublished data ) . this raises the possibility that multiple gefs function during midline guidance and that disrupting just one has limited effect . for example , mutations in drosophila trio , another rho gef with well - established roles in regulating axon outgrowth , cause only minor disruptions in the cns axon scaffold , whereas they have more profound effects in combination with other mutations that affect midline axon guidance ( awasaki et al . , 2000 ; bateman et al . , 2000 ; liebl et al . , 2000 ; newsome et al . alternatively , it is possible that gef64c mutations do cause defects in subsets of cns neurons , but that these defects are not readily apparent when all axons are visualized simultaneously . to further investigate a potential requirement for gef64c in midline axon guidance , we looked at the effects of removing gef64c function in animals that carried mutations in the frazzled ( fra ) gene , which encodes the drosophila homologue of the dcc - attractive netrin receptor ( kolodziej et al . , 1996 ) . mutations in fra cause a range of defects in cns axon guidance consistent with its role in attracting commissural axons to the midline ( fig . fra ; gef64c double mutant embryos exhibit a marked enhancement of the guidance defects typically observed in fra mutants ; there is a substantial reduction in commissure thickness and a greater number of segments where commissures fail to form ( fig . thus , in the fra mutant background where normal axon attraction to the midline is partially defective , loss of gef64c exacerbates these defects , suggesting an endogenous role for gef64c in attractive guidance at the midline . it should be noted that this double mutant analysis does not provide evidence of gef64c 's involvement in dcc signaling , nor does it preclude such a role . dose - sensitive genetic and biochemical interactions between fra and gef64c , which could suggest a direct involvement in dcc signaling , have not thus far been observed . in contrast to the modest effects of loss of gef64c function , pan - neural overexpression of gef64c ( using ep3035 or uasgef64c ) results in a dramatic , dose - dependent , gain of function phenotype , in which many axons abnormally project across the midline . the commissures are thicker and there is a commensurate reduction in the longitudinal axon tracts ( fig . the point mutations in gef64c were introduced on the ep3035 chromosome , allowing for gal4 overexpression of the mutant alleles . none of the mutant alleles , nor the uasgef64cc transgene ( a deletion of the dbl and ph domains ) , were capable of generating the gain of function phenotype , indicating that the abnormal midline crossing is due to gef64c expression , and that this effect requires the intact dbl and ph domains . examination of gain of function embryos with antibodies to wrapper ( noordermeer et al . , 1998 ) , a marker for midline glia , indicates that the guidance defects caused by gef64c overexpression are not a secondary consequence of nonautonomous perturbations of midline glial cell survival or migration ( unpublished data ) . gef64c gain of function overcomes robo repulsion and is suppressed by the rhoa dominant negative . note the more wild - type appearance of commissural and longitudinal axon bundles in the embryo coexpressing gef64c and the rhoa dominant negative ( b ) , relative to gef64c alone ( a ) . the gef64c overexpression phenotype is qualitatively similar to the phenotype of mutations in the robo receptor , raising the possibility that gef64c promotes attraction to the midline by interfering with robo repulsion . first , there are significant differences between the gef64c gain of function and robo loss of function phenotypes : robo mutations have more profound effects on the growth cones that pioneer the ipsilaterally projecting fasii - positive posterior corner cell ( pcc ) pathway than does gef64c overexpression ( unpublished data ) . second , overexpression of gef64c does not appear to affect robo protein expression or localization ( unpublished data ) . the third observation relates to genetic predictions based on the function of commissureless ( comm ) . comm downregulates robo receptors on commissural axons ( tear et al . , 1996 ; kidd et al . , 1998b ) thus , if gef64c overexpression were blocking robo function , the gef64c gain of function should be at least partially epistatic to mutations in comm ; this is not the case ( unpublished data ) . for these reasons , we believe that gef64c overexpression exerts its effects through stimulation of an attractive signaling pathway , rather than through inhibition of robo repulsion . the gef64c gain of function phenotype suggests that by increasing the expression of an attractive signaling molecule , it is possible to overcome the normal repulsive signals that are present at the midline . to determine whether gef64c expression would also allow axons to cross the midline in genetic backgrounds where axons are biased toward being repelled , we coexpressed gef64c with a hyperactive mutant form of the robo receptor : roboy - f ( bashaw et al . , 2000 ) . pan - neural expression of uasroboy - f results in a commissureless phenotype , if in this roboy - f background we simultaneously drive gef64c expression , many commissural axons are now able to cross the midline , and some segments appear to be nearly wild - type ( fig thus , even in this artificially repulsive background , gef64c can allow significant axon growth to and across the midline , raising the exciting possibility that finding ways to stimulate the activity of functionally homologous mammalian gefs could promote regrowth of injured axons in the adult cns . one likely scenario is that it exerts its effects by specifically activating one or more of the rho - family gtpases . there are six rhogtpases in the fly genome : rac1 , rac2 , mtl , rhoa , rhol , and cdc42 ( dickson , 2001 ) . we made use of the gef64c gain of function phenotype and dominant negative gtpase transgenes for rac1 , rhoa , and cdc42 to determine which , if any , of these gtpases are the downstream target(s ) of gef64c , reasoning that genetically limiting the downstream target should suppress the gef64c gain of function phenotype . based largely on the differential effects of rac and rho on neurite extension ( rac promotes extension and rho promotes retraction ) , it has been proposed that during axon guidance rac could play a role in attractive responses , whereas rho could stimulate repulsion ( dickson , 2001 ) . we therefore predicted that the gef64c gain of function phenotype would depend on rac activity , but not on rho . surprisingly , the opposite appears to be true ; the rhoa dominant negative strongly suppresses the gef64c gain of function , whereas the rac1 and cdc42 dominant negatives have little or no effect ( fig . this observation argues against the simplest form of the model that rac mediates attraction , and rho mediates repulsion . to test if the specificity of gef64c for rhoa seen in our genetic experiments is also observed in independent assays for gef64c function , in vitro binding , and guanine nucleotide exchange assays were performed . glutathione s - transferase ( gst ) pull down experiments indicate that gef64c can bind equally well to rac1 , rhoa , and cdc42 ( unpublished data ) , whereas gef64c acts as an in vitro exchange factor for rac and rho ( exhibiting a modest preference in catalyzing the exchange of gdp for gtp on rho , relative to rac ) but does not have exchange activity for cdc42 ( fig . such promiscuity in the in vitro association of gefs with small gtpases has been observed for many rhogefs , including vav and trio ( van aelst and d'souza - schorey , 1997 ) . to further examine the function of gef64c , its effects on the actin cytoskeleton in cultured fibroblasts were determined ( fig . microinjection of a gef64c expression vector into quiescent , serum - starved swiss 3t3 cells resulted in a dramatic stimulation of actin stress fiber formation relative to control cells ( fig . 4 , a and b ) , a phenotype indicative of rho activation ( hall , 1998 ) . coinjection of gef64c and c3 transferase , a protein inhibitor specific for rho ( ridley and hall , 1992 ) , completely blocked gef64c 's ability to induce stress fibers , arguing further that gef64c functions by activating rho ( fig . ( b ) cells injected with a gef64c expression construct show striking actin stress fiber formation . ( b ) injection marker for cells shown in b. ( c ) cells coinjected with the gef64c expression construct and c3 transferase protein . ( c ) injection marker for cells shown in c. ( d ) gef exchange assays for rac , rho , and cdc 42 . the relatively weak , but significant exchange activity that we observe could be attributable to the fact that the ph domain was not included in these assays , as fusion proteins containing both the dbl and ph domains were poorly expressed . the reciprocal loss and gain of function genetic data presented here support a role for gef64c in promoting axon attraction to the cns midline . overexpression of gef64c can overcome the normal repulsive signals present at the midline , and can even drive attraction to the midline in a background where robo repulsion is abnormally strong . surprisingly , genetic and cell culture evidence suggest that these attractive effects are meditated through the activation of rhoa , but not rac . previous evidence from a number of different experimental systems is consistent with the general idea that rac and cdc42 are positive regulators of neurite outgrowth and that rho is a negative regulator ( reviewed in ( luo , 2000 ) ) . these observations on axon outgrowth have been extended to axon guidance , suggesting that rac and cdc42 would mediate attractive guidance responses and rho would mediate repulsion , and have led to the investigation of the role of the rho gtpases in the regulation of axon guidance . for example , ephexin , a gef for rhoa , has been implicated in the repulsive responses mediated by eph receptors ( shamah et al . , 2001 ) , and the repulsive effects drosophila plexin b , a member of the semaphorin receptor family , also appear to be mediated by rhoa ( driessens et al . our findings suggest that the opposite is also possible ; namely , that rhoa may also play a role in attraction , and argue against a single generalizable function for the rho gtpases in axon guidance . the simplest interpretation of our data is that gef64c promotes midline attraction through the stimulation of an attractive signaling pathway . however , it should be noted that until gef64c function is linked to a known receptor / ligand system , it remains a formal possibility that gef64c expression could exert its effects through enhancing the activity of an unknown repellant present in the lateral cns . because such a model would predict strong defects in the longitudinal extension of fasii - positive axons , something that we do not observe nevertheless , discovering how the rho gtpases can elicit different and even opposite axon guidance responses in different contexts is a major challenge for the future , and promises to enrich our understanding of the molecular mechanisms of axon guidance in the developing nervous system . the ep insert in gef64c ( ep3035 ) came from a screen of the 2300 line ep collection provided by the rubin lab . flies from each ep line were crossed to flies that constitutively express the robo - dcc chimera in all neurons : uasrobo - dcc , elavgal4/tm3 . a primary screen was performed for lines that were lethal when coexpressed with the chimera . lines that were lethal independent of the chimera were eliminated , and the remainder were screened anatomically for those that enhanced robo - dcc 's axon guidance defects . ems point mutations in gef64c were isolated in an f1 selection screen for suppressors of the lethal interaction of ep3035 and uasrobo - dcc : ep3035/tm3 males were mutagenized with ems and then crossed to uasrobo - dcc , elavgal4/tm3 . non - tm3 escapers were isolated , retested for suppression , and lines were established . several lines which failed to give the gef64c gain of function phenotype were selected for sequencing . the following fly stocks were also used : ( a ) elavgal4 , ( b ) df(3l)10h , a deficiency for the gef64c region , ( c ) fra / cyowggal , ( d ) comm , ep3035/tm3gal , ( e ) comm , elavgal4/tm3gal , ( f ) uasroboy - f , ( g ) uasracn17 , ( h ) uasrhon19 , ( i ) uascdc42n17 , ( j ) fra / cyowggal ; gef64c / tm6gal , ( k ) fra / cyowggal ; df(3l)10h / tm6gal , and ( l ) ep3035,elavgal4/tm3gal . genomic dna flanking ep3035 was isolated and used to screen the ld embryonic cdna library ( bdgp ) using standard procedures . the full - length gef64c cdna was subcloned into pcdna3.1 ( invitrogen ) and puast for expression in mammalian cells and transgenic flies , respectively . uasgef64cc was derived from the full - length uasgef64c and transgenic lines of each construct were established . the pharmacia pgex system was used for gst fusions of the gef64c dbl and dbl - ph domains : regions of interest were amplified by pcr and subcloned into pgex . gef exchange assays were performed as described previously ( self and hall , 1995 ) . embryo staining and monoclonal antibody production procedures ( gef64c amino acids 13161580 ) were as described previously ( kidd et al . , 1998a ) . quiescent , serum - starved swiss 3t3 cells were prepared as described previously ( nobes and hall , 1995 ) . cells were microinjected with an expression vector ( pcdna3 ) encoding gef64c into the nucleus at a concentration of 200 g / ml . injected cells were marked by coinjection of biotin - conjugated lysinated dextrans ( molecular probes ) at 2 mg / ml , which was detected in fixed cells using alexa 350 streptavidin ( molecular probes ) . rho activity was inhibited by injection of c3 transferase protein with cdnas into the cell nucleus at a concentration of 200 g / ml . after 2.5 h , cells were fixed , without washing , in 4% paraformaldyhyde/0.2% gluteraldyhyde / pbsa for 10 min at room temperature . cells were permeabilized with 0.2% triton x-100/pbs for 5 min and blocked with sodium borohydride ( 0.5 mg / ml in pbs ) for 10 min at room temperature . cells were stained for filamentous actin structures by incubating for 20 min with tritc - conjugated phalloidin ( 0.1 g / ml ) . the ep insert in gef64c ( ep3035 ) came from a screen of the 2300 line ep collection provided by the rubin lab . flies from each ep line were crossed to flies that constitutively express the robo - dcc chimera in all neurons : uasrobo - dcc , elavgal4/tm3 . a primary screen was performed for lines that were lethal when coexpressed with the chimera . lines that were lethal independent of the chimera were eliminated , and the remainder were screened anatomically for those that enhanced robo - dcc 's axon guidance defects . ems point mutations in gef64c were isolated in an f1 selection screen for suppressors of the lethal interaction of ep3035 and uasrobo - dcc : ep3035/tm3 males were mutagenized with ems and then crossed to uasrobo - dcc , elavgal4/tm3 . non - tm3 escapers were isolated , retested for suppression , and lines were established . several lines which failed to give the gef64c gain of function phenotype were selected for sequencing . the following fly stocks were also used : ( a ) elavgal4 , ( b ) df(3l)10h , a deficiency for the gef64c region , ( c ) fra / cyowggal , ( d ) comm , ep3035/tm3gal , ( e ) comm , elavgal4/tm3gal , ( f ) uasroboy - f , ( g ) uasracn17 , ( h ) uasrhon19 , ( i ) uascdc42n17 , ( j ) fra / cyowggal ; gef64c / tm6gal , ( k ) fra / cyowggal ; df(3l)10h / tm6gal , and ( l ) ep3035,elavgal4/tm3gal . genomic dna flanking ep3035 was isolated and used to screen the ld embryonic cdna library ( bdgp ) using standard procedures . the full - length gef64c cdna was subcloned into pcdna3.1 ( invitrogen ) and puast for expression in mammalian cells and transgenic flies , respectively . uasgef64cc was derived from the full - length uasgef64c and transgenic lines of each construct were established . the pharmacia pgex system was used for gst fusions of the gef64c dbl and dbl - ph domains : regions of interest were amplified by pcr and subcloned into pgex . gef exchange assays were performed as described previously ( self and hall , 1995 ) . embryo staining and monoclonal antibody production procedures ( gef64c amino acids 13161580 ) were as described previously ( kidd et al . , 1998a ) . quiescent , serum - starved swiss 3t3 cells were prepared as described previously ( nobes and hall , 1995 ) . cells were microinjected with an expression vector ( pcdna3 ) encoding gef64c into the nucleus at a concentration of 200 g / ml . injected cells were marked by coinjection of biotin - conjugated lysinated dextrans ( molecular probes ) at 2 mg / ml , which was detected in fixed cells using alexa 350 streptavidin ( molecular probes ) . rho activity was inhibited by injection of c3 transferase protein with cdnas into the cell nucleus at a concentration of 200 g / ml . after 2.5 h , cells were fixed , without washing , in 4% paraformaldyhyde/0.2% gluteraldyhyde / pbsa for 10 min at room temperature . cells were permeabilized with 0.2% triton x-100/pbs for 5 min and blocked with sodium borohydride ( 0.5 mg / ml in pbs ) for 10 min at room temperature . cells were stained for filamentous actin structures by incubating for 20 min with tritc - conjugated phalloidin ( 0.1 g / ml ) .
an atrial septal aneurysm ( asa ) is a rare but well - recognized abnormality of uncertain clinical relevance . it is a localized bulging of the inter - atrial septum into either or both atria during the cardiac cycle . asa has been reported as an unexpected finding during autopsy but may also be diagnosed in living patients by echocardiographic techniques.we present a 37-year - old woman with a recurrent atypical chest pain of one - year duration . she was found to have a congenital atrial septal aneurysm on evaluation . the echocardiographic images mimicked a left sided cor triatriatum sinister . she had no other symptoms and had no co - morbidities . there was no audible murmur.asa can mimic cor - triatriatum and echocardiographic examination should be performed from multiple views before any echocardiographic diagnosis is made .	the first report on atrial septal aneurysm ( asa ) was published by lang and posselt in 1934.1 since then , several asa cases and studies have been published in the literature . an asa is a congenital abnormality that is characterized by a localized bulging of the atrial septum into either or both atria during the cardiac cycle . asa formation can be secondary to interatrial pressure differences , but may also be a primary malformation involving the region of the fossa ovalis or the entire septum . asa may be an isolated abnormality but is often found in association with other structural cardiac abnormalities like mitral valve prolapse or atrial septal defects . initially , asa was considered a rare congenital anomaly , but with the advancement of two - dimensional echocardiography , and more recently , broader use of trans - esophageal echocardiography ( tee ) , detection of this abnormality has become easier and more frequent . to the best of our knowledge , the only previous report of an asa mimicking a cor triatriatum sinister was by ramazan and colleagues.2 studies using trans - esophageal echocardiography ( tee ) have shown a prevalence of 2% to 10% for asa.3,4 most asas are clinically silent , found incidentally in 1% of the general population.5 a 37-year - old african female was referred to the cardiology outpatient clinic with complaints of unresolved atypical recurrent chest pain of one - year duration . she was not known to be hypertensive or diabetic and did not smoke cigarettes or drink alcohol . she denied usage of any medications and was not aware of the presence of heart disease among her family members . on general examination , she was a normal young lady with a body mass index of 24 kg / m . the patient has had several investigations including electrocardiograms , chest radiograph and cardiac enzymes , which were all normal . echocardiography revealed a thickened and myxomatous anterior mitral valve leaflet ; however , no definite mitral valve prolapse was seen . in the parasternal long axis view ( plav ) , a membrane was seen across the left atrium ( la ) , extending anteroposteriorly from the base of the anterior mitral valve leaflet to the base of the posterior valve leaflet ( fig . this membrane was not demonstrable within the la in the apical four chamber and apical two chamber views . the apical four - chamber view revealed a hypermobile inter - atrial septum with at least a 12 mm excursion in either direction ( figs . 2 and 3 ) . multiple parasternal long- and short - axis views , an apical four chamber view and modified echocardiographic evaluations including sub - costal views revealed that the membrane bisecting the la in the plav ( described earlier ) was a cross section of the hypermobile inter - atrial septum atrial septal aneurysm ) . we categorized the patient as having a type 5 atrial septal aneurysm ( bi - directional and equidistant to the right as well as to the left during the cardio - respiratory cycle ; see figs . 2 and 3 ) . our patient did not consent to have a tee , so we commenced on prophylactic aspirin 300 mg daily , which was later reduced to a maintenance dose of 75 mg daily . an asa usually involves the area of the fossa ovalis.5,6 it is a localized bulging of the inter - atrial septum and the diagnostic criteria is usually a protrusion > 6 mm into the right atrium ( ra ) or la or both atriums.7 bulging of the entire atrial septum or localized bulging protruding less than 6 mm is not usually considered to be a true aneurysm , since such bulging might be seen in the absence of a true aneurysm.6 furthermore , a small pocket 36 mm long , extending anteriorly and to the left of the limbus fossae ovalis , has been described anatomically in normal subjects.8 an aneurysm affecting the entire interatrial septum has never been documented pathologically . other authors have used a protrusion of the aneurysm > 10 mm beyond the plane of the atrial septum measured by tee.3 our patient had a bulging of at least 12 mm into either atria during different phases of the cardio - respiratory cycle . the pathogenesis of atrial septal aneurysm might be explained by an abnormal structure of the inter - atrial septum , by a change in the normal inter - atrial pressure gradient , or by both.9 atrial septal aneurysm may also result from bulging of the septum primum tissue through the fossa ovalis.5 asa is mostly a benign entity . it could swing into the la , ra , and back to the la or vice versa . a variety of patterns of sac motion have been revealed in the literature.10,11 atrial septal aneurysm motion was determined according to the classification introduced by olivares - reyes and colleagues.4 the aneurysm is classified as type 1 right ( r ) if the bulging is in the ra only , type 2 left ( l ) if the bulging is in the la only , type 3 rl if the major excursion bulges to the ra and the lesser excursion bulges toward the la , type 4 lr if the maximal excursion of the atrial septal aneurysm is toward the la with a lesser excursion toward the ra , type 5 if the atrial septal aneurysm movement is bidirectional and equidistant to both atria during the cardiorespiratory cycle . the association of asa and mitral valve prolapse ( mvp ) suggests that a myxomatous degeneration might be responsible for both abnormalities.12 our patient had a myxomatous anterior mitral valve leaflet ; however , definite prolapse was not detected . inability to obtain consent for a tee makes it impossible for us to confidently exclude mvp . other authors have observed that a developmental abnormality affecting the tissue of the interatrial septum and mitral valve can be supposed.13,14 atrial septal aneurysm is frequently associated with other atrial septal defects ( asds ) , in particular asd - ostium secundum type , and patent foramen ovale ( pfo).15 if it occurs in isolation it usually does not result in any shunting . these defects were not demonstrable in this study but we acknowledge the limitations of the trans - thoracic echocardiography ( tte ) in detecting a small pfo or asd as opposed to a tee , which could not be completed due to lack of consent . tee has been shown to be more sensitive than tte in detecting asas.3 associations between asas and atrial fibrillation has been documented,16 but our patient did not present with palpitations and we did not document any arrhythmia on the resting electrocardiography . asa has been documented to act as an arrhythmic focus , generating focal atrial tachycardia . it also tends to aggravate stasis of la blood flow and predispose individuals to minute la clots and systemic thromboembolisms . some authors have observed that an asa aneurysm may mimic a right atrial cyst or tumor.17 our patient was commenced on anti - platelet therapy without any anticoagulation . this is in keeping with the findings of homma and colleagues,18 who showed that when anticoagulation therapy ( warfarin ) was compared with aspirin in patients with asa and pfo , no significant differences were noted in regard with the appearance of thromboembolic events . the most appropriate treatment therapy for asymptomatic patients with no significant associated cardiac lesions who are amenable to surgery is uncertain . until more is known about the natural history of this lesion , all management decisions are probably best made on an individual basis . asa mimicking a cor triatriatum remains a rare clinical condition that is usually asymptomatic , but may occasionally present with unresolved atypical chest pain . in the investigation of unresolved chest pain , two - dimensional echocardiographic scanning should be performed from multiple windows , and multiple images should be acquired from conventional and modified views before any echocardiographic diagnosis is made . in addition , associated lesions such as mvp are not uncommon in asa and a tee remains an invaluable modality of investigation for accurate evaluation ; however , this should not be a limiting factor to diagnosis especially when consent is not given or in a resource - poor setting .
a 10-year - boy post - operative , post - radiotherapy case of left temporal glioblastoma multiforme ( gbm ) was referred for f-18 flurodeoxyglucose ( fdg ) positron emission tomography / computed tomography ( pet / ct ) to rule out residual / recurrent disease 6 months following completion of therapy . the fdg scan 3 months following therapy had not shown evidence of viable residual or metastatic disease . the present scan showed a tiny focus of abnormal fdg accumulation in the region of the trigone of the left lateral ventricle which was best appreciated on the plain pet image . a correlative c-11 methionine study showed a well defined focus of abnormal tracer accumulation in the region of the left trigone . cect and mri done subsequently proved it to be a subependymal deposit . this case therefore demonstrates the possibility of subependymal deposits in gbm and the need for this possibility to be entertained during interpretation of the fdg study . it also highlights the advantage of labelled amino acids like c-11 methionine for clearly delineating subependymal deposits apart from the advantage for unequivocal interpretation of the pet study in recurrent brain tumors .	both f-18 fluorodeoxyglucose ( fdg ) and c-11 methionine pet / ct studies were done in a treated case of glioblastoma multiforme who was sent for evaluation of recurrence . apart from recurrence at the primary site a subependymal deposit was noted in the trigone of the lateral ventricle which was well demonstrated on the c-11 methionine study as compared to the f-18 fdg study . this case therefore highlights the important role that c-11 methionine can play for evaluating not only recurrence but also metastatic deposits which are a possibility in such high grade tumors . a 10-year - boy post - operative , post - radiotherapy case of left temporal glioblastoma multiforme ( gbm ) was referred for f-18 flurodeoxyglucose ( fdg ) positron emission tomography / computed tomography ( pet / ct ) to rule out residual / recurrent disease 6 months following completion of therapy . the fdg scan 3 months following therapy had not shown evidence of viable residual or metastatic disease . 185 mbq of f-18 fdg was injected intravenously and the patient was rested for one hour followed by the pet / ct acquisition on a discovery ste 16 camera ( ge ) . low dose ct was followed by 3d pet emission scan of the brain for 15 minutes . images were reconstructed by 3d vue algoritm ( ge ) and viewed on a xeleris workstation ( ge ) using the volumetrix protocol . a tiny focus of abnormal fdg accumulation was noted in the region of the trigone of the left lateral ventricle which was best appreciated on the plain pet image [ figure 1-arrow ] . coronal , saggital and transaxial fused f-18 fdg pet / ct images a correlative c-11 methionine study was done the next day 20 minutes following intravenous injection of 740 mbq of the tracer . a well - defined focus of abnormal tracer accumulation in the region of the left trigone [ figure 2-arrow ] was well - appreciated on the mip , plain pet and fused pet / ct methionine images . f-18 fdg uptake can vary greatly but uptake is high in high grade tumors and their metastases . however , because of the high rate of physiologic glucose metabolism in normal brain tissue the detectability of lesions is restricted . amino acid pet tracers like c-11 methionine in contrast have high uptake in tumor tissue and low uptake in the normal brain , thus giving better lesion to background ratios . they are transported into the cell via carrier mediated transport processes and transport is upregulated following malignant transformation . in this case because the subependymal deposit was away from the grey matter it could be visualised on f-18 fdg pet , however this deposit was clearly delineated on the c-11 methionine study and correlated well with the cect and mri findings [ figure 3 ] . gbm is the most aggressive type of brain tumor and has a very poor prognosis . the tumor may extend into the meninges or ventricular wall and malignant cells carried in csf may spread to the spinal cord or cause meningeal gliomatosis . though various studies in literature have compared f-18 fdg with c-11 methionine for evaluation of gliomas and recurrent brain tumors.[68 ] their comparison in metastases such as this subependymal deposit has not been reported . the possibility of subependymal metastases should be kept in mind while reporting the f-18 fdg brain study in gbm and these deposits can be well delineated with amino acid tracer such as c-11 methionine study . coronal , saggital and transaxial fused c-11 methionine pet / ct images ( a ) axial cect done subsequently showing the mildly enhancing subependymal nodule and ( b ) axial flair mr sequence showing the hyperintense subependymal nodule in the trigone of the left lateral ventricle
the thermal aggregation of the biopharmaceutical protein recombinant protective antigen ( rpa ) has been explored , and the associated kinetics and thermodynamic parameters have been extracted using optical and environmental scanning electron microscopies ( esems ) and ultraviolet light scattering spectroscopy ( uv - lss ) . visual observations and turbidity measurements provided an overall picture of the aggregation process , suggesting a two - step mechanism . microscopy was used to examine the structure of aggregates , revealing an open morphology formed by the clustering of the microscopic aggregate particles . uv - lss was used and developed to elucidate the growth rate of these particles , which formed in the first stage of the aggregation process . their growth rate is observed to be high initially , before falling to converge on a final size that correlates with the esem data . the results suggest that the particle growth rate is limited by rpa monomer concentration , and by obtaining data over a range of incubation temperatures , an approach was developed to model the aggregation kinetics and extract the rate constants and the temperature dependence of aggregation . in doing so , we quantified the susceptibility of rpa aggregation under different temperature and environmental conditions and moreover demonstrated a novel use of uv spectrometry to monitor the particle aggregation quantitatively , in situ , in a nondestructive and time - resolved manner .	the study of protein aggregation is a burgeoning field of research driven by the urgent need to elucidate the mechanism of neurodegenerative diseases , the desire to understand and mimic natures ' ability to create hierarchical complex nanostructures , and the necessity to understand and minimise product loss during the processing and formulation of biopharmaceuticals . aggregation is of particular importance for therapeutic proteins as it can lead to a loss of product , reduce efficacy , alter biological activity and pharmacokinetics , and even raise safety concerns such as increased immunogenicity [ 13 ] . aggregation can be induced by solution conditions such as protein concentration , ph , salinity , temperature , and the presence of additives [ 4 , 5 ] . stresses to the protein such as over - expression , refolding , freeze - thaw cycles , agitation , or exposure to hydrophobic surfaces or air ( including foaming ) can also lead to the formation of aggregates [ 2 , 46 ] . each of these environmental factors is typically encountered during bioprocessing , downstream processing , storage , and also during and after in vivo delivery of biopharmaceutical actives . hence , there is significant on going work channelled into exploring the onset of aggregation and the aggregation pathway . understanding these factors subsequently allows the development of an informed strategy to minimise aggregation during biopharmaceutical production , for example , the inclusion of surfactants to influence protein monomer interactions . the influence of solution ph on aggregation is one of the more studied and important parameters that controls the onset of aggregation and final aggregate morphology . this is because ph alters the surface charge of the protein monomer and also the extent of any structural disruption prior to aggregation , and hence influences their propensity to self - assemble and the manner in which they go onto aggregate . for example , at ph values close to the isoelectric point , repulsive interactions between native monomers are reduced , making assembly more favourable . under these conditions , phs far from the isoelectric point , increased charge repulsion within the protein destabilises the folded conformation , leading to the exposure of hydrophobic groups , which in turn can drive the self - assembly of these unfolded states , typically into -sheet rich fibrillar structures [ 8 , 9 ] . temperature is another key factor , if not the most critical factor in commercial processes that induces aggregation , where increasing the temperature increases the vibrational motion and diffusion of proteins which is a necessary step for aggregation . moreover , as the temperature nears the denaturation temperature of the protein , the protein partially unfolds , exposing hydrophobic regions , which induces aggregation . the literature is awash with many studies that postulate different models for the self - assembly of proteins , and these can generally be divided into two main categories : empirical or mechanistic . mechanistic models are based on a reaction scheme and have parameters relating to the kinetics / thermodynamics of the process , whereas empirical models utilise functions that fit the data but have no physical meaning . a large array of different mechanistic models have been proposed , and these have been broadly categorised as monomer addition , reversible association , prion aggregation , minimalistic 2-step , or quantitative structure - activity relationships . alternatively , roberts proposed a comprehensive generic mechanistic scheme for protein aggregation with associated generic equations , and he went onto show how these could be simplified for certain conditions and limiting cases . overall , most of these models are based on the idea of aggregation being mediated by a reactive intermediate which is in equilibrium with the native state ; the intermediate is able to aggregate , initially via a nucleation step followed by a growth phase , that is , native ( n ) intermediate ( i ) aggregate ( a ) . many groups have experimentally tested such models , and techniques have been developed and exploited to study aggregation both ex situ and in situ [ 13 , 14 ] . in ex situ , the state of aggregation is measured in static samples , where long data acquisition times are needed , or samples need to be separated , dried , fixed or labelled , for example , using electron microscopy or mass spectrometry . furthermore , the aggregation process has to be reliably halted and persevered for a series of samples representing different stages . in in situ measurements , aggregation events are monitored as they happen , but the technique used needs sufficient time resolution for the process being monitored and the data analysis can be complex , since there is typically a mixture of component sizes , that is , aggregates and monomers . typical techniques currently used include circular dichroism , fourier transform infra - red , and dynamic light scattering [ 13 , 15 ] . for a technique to yield useful information about the aggregation process , it is crucial to be able to relate the measured parameter back to changes in the aggregation state such as aggregate size or monomer depletion . here , the thermal aggregation of an industrially relevant biopharmaceutical recombinant protective antigen ( rpa ) ( active component in a second - generation anthrax vaccine ) has been examined visually and via turbidity measurements , before information on the size and the shape of the aggregates formed were obtained using a combination of optical and environmental scanning electron microscopies . two environmental conditions were explored , where samples were prepared with and without the denaturant urea . subsequently , the rate of aggregate growth at different isothermal temperatures was compared using ultraviolet light scattering spectroscopy ( uv - lss ) [ 1723 ] , with particle size being extracted as a function of time . this was done by collecting spectra over time without the need to remove material for analysis , hence demonstrating a nondestructive , in situ , and time - resolved method for monitoring the aggregation process . results for the aggregation of rpa will be discussed , and a model describing the kinetics and thermodynamics of aggregation is presented . rpa was supplied by avecia biologics ( uk ) at 2 mg ml in a phosphate - buffered saline solution adjusted to ph 7.4 . doubly distilled water was obtained from an elga purelab ultra ( 18.2 ) . all other chemicals were purchased from either sigma - aldrich ( uk ) or acros organics ( uk ) , where the reagent grade is at least 97% pure and used as recieved . the rpa samples supplied were treated using the following procedure to provide a consistent starting material for analysis . initially , rpa was precipitated from solution by heating ( 50c for circa 5 min ) . the resulting gel was centrifuged at 6000 rpm for 1 minute , and the supernatant was discarded . the gel was resuspended by adding doubly distilled water and vortexing for 2 min . the gel was resolubilized by adding urea and doubly distilled water to form an 8 m urea solution . rpa was refolded by dilution , using 1 part rpa in 8 m urea to 31 parts refold buffer . this was done in two stages , with a 1 : 7 dilution followed by 1 : 4 dilution 2 minutes later . the refold buffer contained 25 mm tris , 25 mm nacl , 2 mm cacl2 and was adjusted to ph 7.4 with hydrochloric acid . the final buffered samples contained between circa 0.15 and 0.3 mg ml rpa with 0.25 m urea and were analysed immediately after refolding . this concentration was chosen as it mimics the conditions of storage of some of the formulations of protective antigen vaccines . samples without urea were prepared using the refolding method followed by dialysis using 3500 dalton molecular weight cutoff visking dialysis membrane ( medicell international ltd ) against 10 times excess of chilled refold buffer for 18 hours in the refrigerator ( ~4c ) . after this , the external solution was replaced twice with fresh refold buffer and allowed to dialyse for a further 24 hours . the rpa sample was then recovered from the sealed membrane and stored in the refrigerator ( circa 4c ) prior to use . the concentration of the refolded rpa solution was determined from its uv absorbance at 280 nm ( shimadzu uv 2501-pc spectrophotometer ) , using a molar absorption coefficient of 72769 m cm . the isothermal aggregation of rpa was monitored visually over time by incubating samples over a range of temperatures ( 4349c ) close to the denaturation temperature of rpa ( 50.0c ) by using a recirculating water bath connected to a heating stage that contained the sample cell . the temperature was measured using a calibrated k - type thermocouple placed in the sample solution ( accurate to 0.3c ) . images of rpa aggregates were obtained using a zeiss axioplan 2 in transmission mode with a 10x magnification objective and a digital camera . the aggregated samples were pipetted onto a microscope slide and a cover slip placed on top . aggregates were mounted for esem analysis simply by lifting them out of solution on a mica disc and placing them directly onto the sample stage . the samples were examined using a philips fei quanta 200 esem with the electron gun accelerating voltage set to 30 kv , the sample stage at 5c , and the chamber pressure at 6 torr . light scattering spectra were recorded using a shimadzu uv 2501-pc spectrophotometer set to record between 250 and 390 nm every 2 nm at a medium scan rate . the wavelength dependence of absorbance arising from light scattering by particles in solution follows the relationship ( 1)a= , where a is the absorbance at wavelength , is a constant and is the scattering exponent . the scattering exponent can be related to particle size using mie theory . here , the mie equations were solved over a range of particle sizes ( nanometres to micrometres ) for wavelengths between 320 and 390 nm using a fortran programme adapted from bohren and huffman . these calculations required the refractive index of the particles and the surrounding solution for all relevant wavelengths . the solution refractive index was taken to be that of water and the particle refractive index was calculated by evaluating the lorentz - lorenz molar refraction using the chemical formula of rpa and a density of 1.43 g cm . the density was based on the molecular weight of rpa ( 82667 da ) and its volume ( 95.74 nm ) as calculated by vadar using the crystal structure of rpa , which is stored in the protein data bank ( http://www.pdb.org/ ) under pdb i d : 1acc . this value of density was considered reasonable compared to the density of other proteins with a similar molecular weight . the values of refractive index , n , calculated for the particles at different wavelengths , , within the visible region are given in table 1 . these values were subsequently extrapolated to the uv region using the cauchy equation ; ( 2)n=1.642 + 79802 . the results obtained using mie theory were analysed to give a theoretical scattering exponent versus diameter , providing a means of converting experimental scattering exponents to particle size : ( 3)d=0.686 6 + 6.869 521.397 4 + 23.795 323.682 2111.896 +642.207 , where d is the particle diameter ( nm ) and is the scattering exponent between 320 and 390 nm . solutions of rpa ( 0.31 mg ml ) were incubated over a range of temperatures ( 2550c ) , and their visual appearance was monitored over time . this temperature range was selected as the denaturation temperature of rpa is known to be ~50c . a typical example of these changes is recorded in figure 1 , which shows the visual appearance of rpa in solution over time whilst held at a steady temperature of 47.6c . initially , the sample was clear , as can be seen from figure 1(a ) , and remained clear over the first few minutes . after 8 minutes , the sample started to become cloudy , which is evident in figure 1(b ) . the slightly cloudy homogeneous appearance of the sample indicates the presence of microscopic particles of sufficient size and concentration to noticeably scatter visible light . this can be seen by comparing the images in figures 1(b ) and 1(c ) ; the latter appears cloudier . after 32 minutes , the sample was no longer homogeneous , since particles large enough to be observed visually started to form ( see figure 1(e ) ) . from this point forwards , the black background was removed to provide improved contrast for observing the macroscopic particles that were forming in the solution ; the sample remained cloudy but appeared brighter , since light was able to enter from behind . the macroscopic particles were more easily observed and appeared as dark spots against the bright cloudy solution . following their emergence , the macroscopic particles were observed initially to increase in size and number as can be seen from the images shown in figures 1(e ) and 1(f ) . beyond this point , the number of individual particles appeared to fall whilst continuing to grow in size ( figure 1(g ) ) . the final image of the sample , figure 1(h ) , was taken with the black background replaced to enable comparison with the initial sample appearance . it shows that the sample had returned to a clear solution apart from the presence of large white particles , some of which had settled on the bottom and the sides of the cuvette . such observations suggest that small microscopic particles form initially , causing the sample to appear turbid , and subsequently cluster to form large macroscopic particles that sediment due to gravity when they are above a critical size . these visual results were complemented by turbidity measurements recorded under identical conditions ( see figure 2 ) ; the labels ( a to h ) in figure 2 are positioned to relate to the photographs taken of rpa aggregation shown in figure 1 . the turbidity was assessed by recording the optical density using light with a wavelength of 320 nm . figure 2 shows a sharp rise in turbidity over the first 20 minutes of incubation . this is consistent with the appearance and growth of particles in the solution , as indicated by the visual observations . the subsequent fall in turbidity suggests that the particles were either decreasing in size ( which might occur if the aggregation was reversible ) or decreasing in concentration ( which could occur if the particles clustered to form larger structures ) . the latter is consistent with our visual observation of particles growing in size and reducing in number over time , before settling to the bottom of the sample cuvette . similar visual and turbidity observations were noted for other samples incubated between 43 and 49c , as all appeared to have a similar two - step mechanism . the time scales for each step varied widely ; however , for the different temperatures , the time to reach peak turbidity ranged from circa 10 minutes at 49c to 10 hours at 43c . this dramatic increase in aggregation rate versus temperature could be driven by either increasing the translational kinetic energy of protein monomers , causing more frequent collisions between the particles , or increasing the internal kinetic energy of the protein monomers . the latter will drive monomers toward an unfolded state , with the associated exposure of hydrophobic patches increasing the likelihood that a collision results in self - association . the morphology of the macroscopic particles formed after incubation was examined using optical and electron microscopy . figure 3(a ) shows a typical optical micrograph of the rpa aggregates formed after 16 hours at 47c . the micrograph reveals the formation of several aggregates with different shapes and sizes , but all appear to be composed of microscopic particles . this is consistent with the visual observation of a cloudy solution of microscopic particles which cluster to form large aggregates . the aggregates generally range in length from ~75 to 730 m and have a width of ~5150 m . three aggregates with fairly distinct structures have been labelled , , and in figure 3(a ) . the smallest , labelled , is 165 m long and 30 m at its widest point . this aggregate appears reasonably linear with growth predominantly in one direction with limited side branching . the other two aggregates , and , are larger and have similar dimensions : ~380 m by 150 m . however , has a very dense structure , whilst has a more open structure . the open structure of consists of a branched system of linear components similar to aggregate , suggesting that is a precursor to the formation of : either as one of many components that come together or as the starting point for further growth or a combination of both . the structure of is densely packed ; however , there is still some evidence of a branched structure and these are similar to the structural features observed in . suggesting that is possibly a precursor to the formation of . such observations and inferences are also supported by previous work on fractal aggregates , reviewed by meakin . such comparisons suggest that the rpa aggregates described here are fractal aggregates , confirming that they are formed by the clustering of microscopic particles . to obtain further information on the size and the shape of the microscopic particles , the aggregates were viewed under esem and a typical micrograph is given in figure 3(b ) . the microscopic particles appeared reasonably spherical and uniform with a diameter of approximately 500 nm . this observation is consistent with the previously reported results for the thermal aggregation of proteins close to their isoelectric points , which showed the formation of monodisperse particulates . here , we are working at ph 7.4 , which is close to the isoelectric point of rpa ( ph 5.6 ) ; therefore , individual protein monomers will have reduced net charge . in addition , the salt present in the buffered media will screen any remaining charge on the proteins , thus any long - range charge - charge repulsions which could act as a barrier to aggregation will have been minimised . the protein monomer is likely to be partially unfolded as the temperature approaches the denaturation temperature ( ~50c ) . the exposed hydrophobic regions will consequently drive nonspecific monomer aggregation under these conditions of reduced net charge . this in turn leads to the formation of the three - dimensional spherical aggregates , as observed in figure 3(b ) . the approximately uniform size of the particles in each sample suggests that their concentration remained reasonably constant during their growth which is consistent with the features of protein aggregation summarised by gosal and ross - murphy . a slight increase in particle diameter from 360 to 500 the rate of aggregation at different isothermal temperatures was explored using uv light scattering spectroscopy ( uv - lss ) , where spectra were recorded every 20 seconds initially and then every 20 seconds to 40 minutes depending on the aggregation rate . such fast acquisition times provide a speed advantage over other light scattering techniques for analysing such aggregation kinetics . it is clear that there was no absorbance initially ( no chromophores in the protein absorb over this wavelength range ) , but the absorbance intensity increases over time , where the difference between successive spectra is large initially but reduces over longer times this implies that the aggregates grew quickly initially when the rpa monomer concentration was at its highest , followed by slowing growth as the rpa monomers were consumed and falling in concentration . as discussed previously , rpa solutions became turbid during incubation at temperatures 43c ; therefore , it can be assumed that multiple light scattering was occurring . it can be assumed , however , that the wavelength dependence of light scattering obtained by solving the mie equations would hold for the scattering occurring here . as such , the uv spectra in figure 4(a ) were analysed ( see section 2.8 ) to give aggregate diameter versus time ; see figure 4(b ) . it is clear that the particles grew quickly over the initial ~60 min before gradually slowing and reaching a final particle size after ~300 min at this temperature . the equilibrium sizes of particles formed increased slightly from 390 to 500 nm with increasing the incubation temperature , which correlates well with the esem observations ( ~360500 nm ) . this confirms our assumption that the wavelength dependence of light scattering in a turbid solution can be adequately approximated by mie theory . to explore the effect of isothermal temperature on particle growth rate and consequently gain an insight into the aggregation kinetics , particle size was recorded as a function of time for a range of temperatures ( 4349c ) and the results are given in figure 5 . the aggregate diameter versus time profile was fitted with ( 4)dmodel = dfd0et/ , where dmodel is the model fit to the particle diameter ( nm ) , df is the final particle diameter that the function converges on ( nm ) , d0 is a fitting parameter ( nm ) , is a time constant for the particle growth process ( s ) , and t is time ( s ) . the optimum fit was found using newton 's method to maximise the correlation coefficient , r , whilst allowing the value of df to vary . the values of d0 , , and correlation coefficient were calculated from a straight line fit ( using least squares regression ) to a plot of ln(df dmodel ) versus time . all profiles show an increase in diameter over time ; however , the time taken to reach the maximum diameter is markedly different for each temperature : the higher the temperature , the shorter the time scale to reach the maximum diameter . this is reflected in the results by a notable increase in the profile gradient when the temperature is increased , where the maximum gradient increases by 2.7 times on average for every 1c increase in temperature . similar results were obtained for a range of samples containing an additive : 0.25 m urea . in this case , similar trends were observed over time for a range of incubation temperatures , and the only difference arising was in the rate of particle growth being slightly faster for each sample in the presence of 0.25 m urea . all samples contained a homogeneous distribution of particle size ( confirmed by esem ) suggesting that the aggregate concentration remained constant over time . this means that the quantity of rpa in the aggregates can be determined by estimating the aggregate concentration and using the ratio of aggregate to monomer volume . from this , the difference between the quantity of rpa incorporated within aggregates and the quantity of monomer present initially gave monomer concentration over time , providing a possible means of assessing the aggregation kinetics . to this end , data over initial incubation times ( where the scattering was increasing ) were analysed using a generalised scheme of the protein aggregation pathway . this scheme is outlined in figure 6 , where n is the native state , i is the protein monomer in an intermediate ( unfolded or denatured ) conformational state , aj is an aggregate consisting of j protein molecules , ( am)n is a cluster of particulate aggregates , and k1 to k5 are the rate constants for the different processes . this aggregation pathway can be simplified based on the aforementioned experimental observations ; it was considered reasonable to exclude step d , since the spherical nature of the aggregates inferred that their growth was dominated by single monomer addition rather than clustering to form irregular structures , and step b was also considered not to have played a significant role , since the aggregates formed were reasonably uniform in size , for which the aggregate concentration would have had to be reasonably constant during their growth . it is reasonable to expect that if new aggregates had formed throughout the aggregation process , then a wide distribution of aggregate sizes would have been observed . the modelling of the aggregation kinetics was further simplified by considering two limiting cases : unfolding limited aggregation and association - limited aggregation . both were tried and association - limited aggregation was found to be the most appropriate as it gave a better fit to the experimental data and more reasonable kinetic values in comparison to previous work [ 33 , 34 ] . in the case of association - limited aggregation , step a is more rapid than step c , ( k1 and k2k4 ) . as such , n and k2ci . the total monomer concentration , cm , is the sum of the concentrations of the monomers in the native state ( n ) and the structurally altered state ( i ) , that is , cm = cn + ci . combining these two relationships gives ( 5)cm=(1+k)cik , where k is the equilibrium constant , k = k1/k2 . since step c is the rate limiting step , the resulting kinetic model is a second - order rate equation , which incorporates the equilibrium constant in order to be stated in terms of cm , ( 6)(rm)=k4cmcak(1+k ) . as stated previously , the aggregate concentration , ca , is expected to have been reasonably constant ; therefore , the model can be reduced to pseudo - first order ( 7 ) , where the equilibrium constant is incorporated into the rate constant for the rate equation ( 8) as follows : ( 7)(rm)=kpseudo cm,(8)kpseudo = k4cak(1+k ) . this model was subsequently used to estimate the aggregation kinetics of all samples using the experimental particle diameter , d(t ) , ( figure 5 ) to calculate the number of monomers in each aggregate as a function of time ( an(t ) ) from the ratio of the monomer volume to aggregate volume via ( 9)an(t)=[d(t)]36vm . the volume of a pa molecule ( vm ) was taken to be 95.74 nm , which was obtained using the crystal structure , as described previouslt . the free monomer concentration versus time ( cm(t ) ) was obtained by taking the difference between the initial monomer concentration ( cm0 ) and the amount of monomers in the aggregates , based on the previous assertion that the aggregate concentration remained constant , ( 10)cm(t)=cm0[caan(t ) ] . the final size of aggregate that the function converges on is assumed to be the size where aggregates would have stopped growing if the clustering process is not interfered with the particulate growth phase . as such , this is the point at which all the protein monomers would have been consumed and all the proteins would have been present in the form of the particulate aggregates . the aggregate concentration is therefore given by ; ( 11)ca = cm0 6vm ( df)3 . this procedure was used to generate concentration versus time profiles for the best fit pseudo first order kinetics over a range of temperatures , and the results for the 0.25 m urea samples are shown in figure 7(a ) , and the values for the second - order k 's are provided in figure 7(b ) . it is evident that in each case the fitted data are in good agreement with those obtained experimentally and the magnitudes of the rate constants obtained from these data are reasonable , although a little high , compared to those reported elsewhere for the association - limited aggregation of protein ( e.g. , bovine granulocyte colony - stimulating factor ) ; extracted values were in the region of 10 to 10 dm mol s over a similar temperature range . it is clear from figure 7(b ) that the association - limited aggregation rate constants for samples with and without added urea increase exponentially with temperature . on average if the data are re - plotted as lnk versus 1/t ( figure 7(c ) ) , it is clear that the data follows arrhenius ' law , k = afexp(eact / rt ) , where af is the pre - exponential factor , t is the absolute temperature ( k ) , eact is the activation of the reaction ( j mol ) and r is the ideal gas constant ( j mol k ) . from the gradients of each slope the activation energies for the two sample types were calculated to be 942.0 92.4 kj mol for rpa without additives and 928.8 32.5 kj mol for rpa with 0.25 m urea . these values suggest that the addition of urea reduced the activation energy slightly , which correlates with the observation that the presence of urea increases the rate of aggregation . the magnitude of these activation energies are reasonable compared to those reported elsewhere for the association limited aggregation of protein ; reported as being between ~420 and ~840 kj mol . these values are reported as observed activation energies and are accompanied by the suggestion that the temperature dependence of association limited rate constants does not follow true arrhenius behaviour . the frequency factors , af , for the two sample types were calculated to be 1.8 10 dm mol s for rpa without additives and 4.7 10 dm mol s for rpa with 0.25 m urea . these values are very high and unlikely to have any physical significance . published values of the second - order frequency factors for various small molecule solution phase reactions are in the range of 10 to 10 dm mol s . this suggests that the arrhenius equation is no more than an empirical fit to the data here . this is not unexpected given the suggestion cited previously that the temperature dependence of association - limited rate constants does not follow true arrhenius behaviour . however , the correlation coefficients between the data and the lines of the best fit show a reasonable fit : 0.963 for the samples without additives and 0.973 for the samples with 0.25 m urea . an alternative and more appropriate approach to modelling the temperature dependence of the rate constant is to factor in the behaviour of the equilibrium constant , k.the analysis using the association - limited model has so far yielded an observed second - order rate constant for the aggregation process . the kinetic model for this analysis is represented by ( 12)(rm)=kobs cm ca , where kobs is the observed rate constant . comparing this with ( 6 ) reveals how kobs is related to the actual rate constant ( k4 ) and the equilibrium constant k , ( 13)kobs = k4k(1+k ) . the temperature dependence of k is given by ( 14)ln(k)=srhrt , where s is the entropy change ( j mol k ) , h is the enthalpy change for the process ( j mol ) , t is the absolute temperature ( k ) , and r is the gas constant ( j mol k ) . to fit the experimental data to the temperature dependence of the equilibrium constant , it was assumed that the rate constant , k4 , is independent of temperature . the physical significance of this is that the activation energy is assumed to be negligible for the process of perturbed monomers being added to the growing aggregate . this is a reasonable assumption , since the activation energies for the reaction of highly reactive free radicals can be close to zero [ 35 , 36 ] , and given that the perturbed monomers will have highly unfavourable hydrophobic patches exposed to water , they too are likely to be highly reactive and easily associated with an aggregate in order to reduce their free energy . rearranging ( 13 ) and equating it to ( 14 ) yields ( 15)ln(kobsk4kobs)=srhrt . therefore , plotting ln(kobs/(k4 kobs ) ) versus 1/t will yield a straight line , the gradient of which will be h / r and the intercept s / r . the initial estimate for k4 was picked by choosing a value greater than the largest value of kobs so that the logarithmic term could be satisfied . when the 0.25 m urea data was plotted , it was found that the correlation coefficient for the fit between the line of the best fit and the data points improved as the value of k4 was increased . it was on this basis that an optimum value of 5.77 10 dm mol s was selected for k4 . the value of k4 effectively represents the value of the frequency factor , since the activation is assumed to be zero . as such , this optimum value of rate constant / frequency factor is much more likely to have a physical significance , since it is comparable to general values of the frequency factor found in the literature discussed previously . the same value of k4 was used for the analysis of both sets of data : rpa without additives and rpa with 0.25 m urea . this was done on the assumption that the collision rate would not be significantly altered in the presence or the absence of urea . it is clear that the correlation coefficients between the data and the lines of the best fit are in reasonable agreement for both sets of samples . h and s were extracted from the graph for each sample and used to calculate the gibbs free energy change , g , at 25c . the values obtained ( table 2 ) were compared reasonably well with those reported in the literature for the g between folded and unfolded proteins , reported to typically be between 20 and 60 kj mol . these values also indicate , as expected , that the presence of urea reduces the stability of rpa , most likely by helping to disrupt the native structure of the protein , causing it to become perturbed at temperatures lower than those in the absence of any urea . to explore the effect of temperature on the extent of disruption of individual proteins , the fraction of protein in the perturbed intermediate state , x , was calculated using ( 16 ) and plotted as a function of temperature ( figure 9 ) : ( 16)x = cicm = k(1+k ) . extrapolation of this data to ambient temperatures shows that the fraction of protein in the perturbed state is negligible at low temperatures . for example , extrapolation to 25c for the sample with 0.25 m urea shows that the fraction of protein in the perturbed state is 10 . this corroborates our experimental observations that rpa does not show observable aggregation over extended periods ( circa 16 hours ) at ambient temperature . the unfolded fraction has also been extrapolated to higher temperatures , using ( 11 ) and ( 12 ) , and the results have been shown by the extrapolated line of the best fit in figure 9 . the inset of figure 9 shows that the fraction of monomer in the perturbed state rises sigmoidally between 46c and 55c and that a higher proportion of protein is perturbed when urea is present . both observations correlate with the susceptibility of the protein samples to aggregate , that is , the more protein perturbed , the higher the chances and the faster the kinetics of aggregation . for example , 8-anilino-1-naphthalene sulfonate ( ans ) and rpa reported elsewhere show that the hydrophobicity of rpa increases sigmoidally versus the temperature between 40 and 55c , as was also the case when the thermal unfolding was examined by circular dichroism , which revealed a sigmoidal increase in unfolding versus the temperature between 45 and 55c . the susceptibility of a biopharmaceutical protein rpa to aggregate as a function of temperature and formulation conditions has been determined , and the kinetics and thermodynamics of the aggregation process have been modelled and quantified . visual and turbidity experiments showed that the thermal aggregation of rpa occurs at incubation temperatures 43c , which is close to its denaturation temperature . under these conditions , the protein is likely to have increased translational kinetic energy , hence more collisions will take place , and also be at least partially unfolded , hence , it has some exposed hydrophobic regions which are known to induce rapid and nonspecific aggregation . such aggregation was found to proceed in a stepwise manner , by first forming spherical microscopic particles followed by clustering to form fractal aggregates . increasing the temperature more than 43c increased the rate of aggregation dramatically and also the size of the diameter of the spherical microscopic particles formed from ~360 to 500 nm when increasing the temperature from 43 to 49c . we went on to show that the growth of the microscopic particles can be monitored using uv - lss . in particular , we used the increase in scattered light from the sample over time to elucidate aggregate size versus time , giving a quantitative measure of the aggregation . moreover , the experiments were conducted over a range of temperatures , with and without 0.25 m urea , and as such , the results were analysed to determine the rate constant and the temperature dependence of the thermal aggregation process . based on this analysis , we proposed that the aggregation process is association limited and that the temperature dependence relates to the equilibrium behaviour between native and perturbed states . we were also able to extract the thermodynamic parameters for aggregation in samples with and without urea , and these indicated that the presence of urea reduces the stability of rpa , hence increases its susceptibility to aggregation . the modelling tools developed here for analysis of data from the easily accessible uv - lss technique provides a fast between in situ analysis method for comparing the stability of different formulations of protein when exposed to different environmental conditions . this method has an important speed advantage over other light scattering techniques when analysing such particle growth kinetics . this work , therefore , provides a basis for quantitatively exploring the effect of additives and/or different processing conditions on the rate of aggregation of industrially relevant biopharmaceuticals with the aim of minimising any self - association during production , downstream processing , or storage .
arteriovenous malformations of jaw are extremely rare conditions that can result in disastrous complications , if handled carelessly . although various treatment modalities have been advocated in the literature , there seems to be no complete consensus on a suitable treatment in these cases . this report highlights the importance of correct diagnosis and early treatment in management of vascular malformations . extracorporeal curettage followed by immediate replantation yielded good results in our case and this technique can emerge as a valid alternative , especially in developing countries .	. the lesion can present with a variety of clinical findings depending on the severity of malformation . interosseous avm often remains undiagnosed until a dramatic bleeding incident occurs , which is usually a result of dental manipulation . various treatment modalities have been described for treatment of high flow avm , most of them should be adjunct to surgery . curettage of the resected fragment with immediate replantation reduces the morbidity associated with the procedure and the difficulty of reconstruction . this seems to be a good approach . following the above logic , here we present a case where avm of mandible was treated with extracorporeal curettage of lesion and immediate replantation of segment . a 16-year - old female patient reported to emergency department with a massive bleeding episode after extraction of tooth , which could only be controlled after biting on gauze piece . bleeding stopped only after applying sustained local pressure over the extraction site . history revealed that left mandibular second premolar was carious and associated with persistent pain and swelling in the region for the past 2 years . when swelling did not subside even after taking antibiotics , local dentist extracted the tooth without taking any radiographs , resulting in massive uncontrolled bleeding . on detailed examination after 12 hours , a soft , pulsatile , tender swelling with audible bruit the overlying skin was normal in color and there was no paresthesia of the involved region . marginal gingival bleeding was also noted i.r.t 33 , 34 , 36 , 37 with buccal expansion of mandible . rest of the physical examination was unremarkable and other blood investigations were normal . at this stage , the patient refused treatment and returned after few months ; meanwhile , swelling had increased to the present size and the patient had suffered from two bleeding episodes . soft , tender , pulsatile swelling in left mandibular region colored ultrasonography of soft submental swelling revealed high flow lesion with multiple feeders . orthopantmograph ( opg ) showed extrusion of 33 , 34 with displacement and tilting of adjacent teeth . the ill - defined moth eaten type radiolucency involved left mandibular parasymphysial body region ; also inferior alveolar canal was dilated and tortourous with enlarged mental foramen [ figure 2 ] . axial , coronal sections of computed tomography ( ct ) scan revealed multiseptae central bony destructive lesion with expansion of bicortical plates without perforation [ figures 3a , b ] . needle aspiration from some distance away from the lesion resulted in syringe full of bright red blood . preoperative opg showing moth eaten irregular radiolucency in left mandibular parasymphysis body region with enlarged and torturous mandibular canal ; adjacent teeth are displaced axial ct and coronal ct section demonstrating widespread destruction and thinning of cortical plates . the patient was taken to the operating room and elective tracheostomy was done to maintain airway postoperatively in case of massive swelling . dissection was done to identify common carotid artery . after achieving proximal and distal control , osteotomy cuts were given in buccal cortical plates away from the lesion , and using bone spreader , inferior alveolar artery was identified and ligated . using extracorporeal technique , teeth were extracted in the involved segment , followed by curettage and hollowing of mandible with curretes , rotating handpiece and bur [ figures 4 and 5 ] . stabilization of reimplanted mandibular piece was done with three 2.5 mm miniplates [ figure 6 ] . follow up of 2 years , ultrasound revealed no new vascular lesion in the region while excellent facial form and function was maintained [ figures 7 and 8 ] . resected mandibular segment containing focal lesion extracorporeal technique of extraoral removal of teeth and curettage of lesion reimplantation of mandibular segment and stabilization with miniplates postoperative opg showing good alignment of mandibular lower border and fixation with plates at symphysis and angle postoperative radiograph showing maintenance of normal anatomical contour the patient was taken to the operating room and elective tracheostomy was done to maintain airway postoperatively in case of massive swelling . dissection was done to identify common carotid artery . after achieving proximal and distal control , osteotomy cuts were given in buccal cortical plates away from the lesion , and using bone spreader , inferior alveolar artery was identified and ligated . using extracorporeal technique , teeth were extracted in the involved segment , followed by curettage and hollowing of mandible with curretes , rotating handpiece and bur [ figures 4 and 5 ] . stabilization of reimplanted mandibular piece was done with three 2.5 mm miniplates [ figure 6 ] . follow up of 2 years , ultrasound revealed no new vascular lesion in the region while excellent facial form and function was maintained [ figures 7 and 8 ] . resected mandibular segment containing focal lesion extracorporeal technique of extraoral removal of teeth and curettage of lesion reimplantation of mandibular segment and stabilization with miniplates postoperative opg showing good alignment of mandibular lower border and fixation with plates at symphysis and angle postoperative radiograph showing maintenance of normal anatomical contour international society for the study of vascular anomalies has classified them as hemangioma ( with endothelial proliferation ) and vascular malformation ( vm ) ( with normal endothelial tumor ) relying on 1982 biologic classification of mulliken and glovacki based on endothelial characteristics . the vms can be further categorized as low flow lesions ( capillary , venous , lymphatic malformations ) and high flow lesions ( avms , arteriovenous fistulae ) according to blood flow characteristics . intraosseous vms of the maxillofacial region sometimes give rise to dental emergencies and may cause disfigurement , morbidity and even death . the proximity of the teeth can prove disastrous , like in our case where extraction of tooth resulted in massive hemorrhage . a review of fatal cases by lamberg and others shows that in most instances , exsanguination is the result of dental extractions , the dentist being unaware of existence of the avm . vascular lesions of the jaws have an overall 2:1 female : male occurrence , with peak incidence in the second decade . in this female patient of 16 years of age , although more than 50% of vascular lesions occur in the head and neck region , only a small percentage of these occur in jaws.[810 ] they are twice as common in the mandible as in the maxilla . mandibular vm usually appears during adolescence , with extremes at 3 months and 74 years of age . vms are caused by a disturbance in the late stages of angiogenesis ( truncal stage ) and result in the persistence of av anastomosis present during embryonic life . vms , which usually present as developmental anomalies from birth , develop in proportion to physical growth . the increase in size of these vms is asymptomatic and imperceptible at an early age and is promoted by local hemodynamic factors . the blood shunted to the malformation causes the lesion to grow , which in turn causes increased shunting of the blood , hence leading to a vicious cycle . as has been seen in this lesion , it can present with a variety of clinical manifestations depending on the severity of the malformation ; however , occasionally asymptomatic cases have also been reported in patients with av malformation . some of the signs and symptoms reported are soft tissue swelling , paresthesia , pain of variable intensity,[1620 ] teeth mobility and migration , discoloration of overlying skin and intraoral mucosal surfaces , facial asymmetry , local pulsation , noticeable bruit , erythematous gingival and bleeding around the teeth and bone resorption with palpable thrill as well as resorption of the roots in the affected area with no evident tooth - related cause or periapical pathoses . systemic findings like blurred vision , epistaxsis , paresthesia and cardiac abnormalities ( murmur , hypertrophy and failure ) have been reported . the radiographic appearance is quite variable and therefore unreliable as a sole basis of diagnosis . gelfand has summarized three typical radiographic appearances : a sunray appearance created by trabecular bone between the vessels and osetolytic lesion.a soap bubble or honeycomb appearance with occasional punched out areas.an appearance described as ill - defined radiolucency . a sunray appearance created by trabecular bone between the vessels and osetolytic lesion . a soap bubble or honeycomb appearance with occasional punched out areas . an appearance described as ill - defined radiolucency . other radiographic findings may include cortical expansion as well as unilocular or multilocular cystic areas . phleboliths , root resorption and lack of lamina dura have also been described . according to stafne numerous treatments in varying combinations and various degrees of success have been employed , including ligation , embolization , radical resection , use of sclerosing solutions , curettage and packing , radiation , bone wax packing in cavities followed by curettage , in two cases observation and even cryosurgery . embolization reduces blood flow , allowing excision to be performed subsequently within 48 hours2 weeks . embolization is also not without risk as embolic complications , allergic reactions , avascular necrosis of bone , delayed root development , defective mandibular growth have been reported . resection of mandible can result in a variety of disabilities including impairment of speech articulation , salivary control , difficulty in swallowing , trismus and deviation of mandible toward the surgical side during functional movement . this led to the concept of immediate reconstruction which was introduced by weaver et al . who used the patient 's own prefrozen mandibular bone . greene et al advocate filling of particulate marrow in the cavity . as digital subtraction angiography and embolization treatment modality are not available in our institute , we had to rely on traditional method of careful ligation of feeder vessel to prevent excessive blood loss , and intraoperative blood infusion was done to prevent any complications . this young female patient was followed up for almost 2 years and a good esthetic , facial symmetry and functional status was maintained . we have arrived at similar conclusion that this technique is safe , convenient and effective alternative to treat such vascular malformations and restore near exact form , function and symmetry without obviating the need for space maintainers , bone harvesting , future major reconstructive operations and keeping cost factor in check . resection of involved portion followed by curettage and reimplantation can emerge as a valid alternative to secondary reconstructive surgery .
objective : to assess the results of an initial round of supplemental screening with hand - held bilateral breast ultrasound following a negative screening mammogram in asymptomatic women with dense breast tissue who are not at high risk for breast cancer.materials and methods : a retrospective , health insurance portability and accountability act compliant , institutional research board approved study was performed at a single academic tertiary breast center . informed consent was waived . a systematic review of the breast imaging center database was conducted to identify and retrieve data for all asymptomatic women , who were found to have heterogeneously dense or extremely dense breast tissue on screening bilateral mammograms performed from july 1 , 2010 through june 30 , 2012 and who received a mammographic final assessment american college of radiology 's ( acr ) breast imaging reporting and data system ( bi - rads ) category 1 or bi - rads category 2 . hand - held screening ultrasound was performed initially by a technologist followed by a radiologist . chi - square and t - test were used and statistical significance was considered at p < 0.05.results:a total of 1210 women were identified . of these , 394 underwent the offered supplemental screening ultrasound . bi - rads category 1 or 2 was assigned to 323 women ( 81.9% ) . bi - rads category 3 was assigned to 50 women ( 12.9% ) . a total of 26 biopsies / aspirations were recommended and performed in 26 women ( 6.6% ) . the most common finding for which biopsy was recommended was a solid mass ( 88.5% ) with an average size of 0.9 cm ( 0.51.7 cm ) . most frequent pathology result was fibroadenoma ( 60.8% ) . no carcinoma was found.conclusion:our data support the reported occurrence of a relatively high number of false positives at supplemental screening with breast ultrasound following a negative screening mammogram in asymptomatic women with dense breast tissue , who are not at a high risk of developing breast cancer , and suggests that caution is necessary in establishing wide implementation of this type of supplemental screening for all women with dense breast tissue without considering other risk factors for breast cancer .	the sensitivity of screening mammography is decreased by the presence of dense breast tissue , as defined by the american college of radiology 's ( acr ) breast imaging reporting and data system ( bi - rads ) . published studies on hand - held breast ultrasound as a supplemental test to screening mammography in women with dense breast tissue report an incremental cancer detection rate of approximately 24/1000 examined women . breast cancers detected by supplemental ultrasound have been reported to be small invasive cancers , with a high proportion of node - negative cases . however , these studies have important differences in methodology , including varied inclusion criteria and varied qualification of ultrasound performers . recently , and mainly as a result of efforts by grassroots advocacy groups , several states in the united states ( us ) have enacted legislation requiring that , following screening mammography , all women with dense breasts be informed of their breast tissue density and that supplemental screening tests , such as breast ultrasound , should be discussed with them by their providers . in the us , this would entail supplemental screening of more than 40% of women over 40 years of age . the purpose of our study was to retrospectively assess the results of an initial round of supplemental screening with hand - held bilateral breast ultrasound performed consecutively by a technologist and a radiologist following a negative bilateral screening mammogram in asymptomatic women with dense breast tissue who were not at a high risk of breast cancer , as defined by the american college of radiology ( acr ) and the society of breast imaging ( sbi ) . a retrospective , health insurance portability and accountability act ( hipaa)-compliant and institutional research board ( irb)-approved study was performed . a systematic review of the breast imaging center 's database was performed to identify all asymptomatic women who were reported to have heterogeneously dense [ figure 1a and b ] or extremely dense [ figure 2a and b ] breast tissue , as defined by the bi - rads atlas , on screening bilateral mammogram performed from july 1 , 2010 through june 30 , 2012 and who received a final assessment birads category 1 , negative or birads category 2 , benign . ( a ) bilateral mammogram cranio caudal view - heterogeneously dense breasts ( b ) bilateral mammogram medio lateral oblique view - heterogeneously dense breasts ( a ) bilateral mammogram cranio caudal view - extremely dense breasts ( b ) bilateral mammogram medio lateral oblique view - extremely dense breasts at the time of the study , the breast imaging center was initiating a policy by which a paragraph was added to the radiologist 's mammogram report to the referring physician of women meeting the above criteria , stating given the dense breast tissue , which may lower the sensitivity of mammography , supplemental screening with breast ultrasound is offered . at our facility , women at high risk for breast cancer ( 2025% or greater lifetime risk of breast cancer ) , as defined by the american cancer society , are recommended to undergo supplemental screening with breast magnetic resonance imaging ( mri ) , and thus were not included in this study . further , not included were women with a personal history of breast cancer , who at our institution undergo diagnostic , not screening mammography , for life . the following data was retrieved for all women included in the study : age , race , family history of breast cancer , personal history of breast biopsy , personal history of biopsy - proven high risk lesion of the breast , age at menarche , age at menopause , parity , age of first pregnancy , and use of hormones ( hormonal contraceptives and hormone replacement therapy ) . in addition , for women who received the offered breast ultrasound , the following data was also retrieved based on the bi - rads atlas : final assessment bi - rads category of the breast ultrasound exam ; if biopsy was recommended , descriptors of ultrasound findings ( solid vs cystic , measurement of longest axis in cm , shape , margins , echogenicity , posterior acoustic features , orientation ) ; and pathology results if biopsy was performed . further , if the final assessment category of the initial screening breast ultrasound was bi - rads 3 , the results of the first two consecutive short - term follow - up ultrasounds were recorded . an interval follow - up increase in size of a mass by more than 20% in the longest axis was considered to warrant biopsy . every screening bilateral mammogram was obtained as 2d digital study on a selenia- hologic unit and was performed by one of six mammography technologists , all of whom are certified in mammography ( registered by the american registry of radiologic technologists ) , with an experience of 17 , 11 , 6 , 6 , 5 , and 4 years , and was interpreted by one of the four board certified breast imagers , with 30 , 11 , 3 , and 2 years of experience in breast imaging . every supplemental bilateral breast ultrasound examination was obtained less than six months from the screening mammogram and was obtained on a dedicated breast ultrasound unit ( ge logic e9 ) with a high - resolution linear - array transducer ( 615 mhz ) by one of the six technologists , two of whom are rdms certified ( registered diagnostic medical sonographer ) and one of whom is arrt certified ( american registry of radiologic technologists ) . two of them with experience in breast sonography for 10 years each , one with 6 years , two with 5 years of experience each , and one with 1-year experience . the breast ultrasound was performed in a standardized hand - held manner with overlapping scans in the radial and antiradial planes , extending from the nipple to the posterior breast tissue . if no abnormal findings were identified , images were documented in the 12- , 3- , 6- , and 9-oclock positions , as well as in the retroareolar region . if any finding was present , images of each finding were obtained and measured in three dimensions . immediately after the breast ultrasound exam by the technologist , the exam was repeated by one of the four board - certified dedicated breast imaging radiologists . the breast imager performed a repeat complete ultrasound scan of the breasts in real time , regardless of whether or not the technologist identified any abnormality and was not blinded to the results of the screening mammogram and the patient 's history and was able to review the breast ultrasound images obtained by the technologist . all ultrasound - guided breast biopsies were performed either with a 14-gauge automated core biopsy needle ( achieve , cardinal health , dublin , ohio ) , or a 9 or 12-gauge vacuum - assisted core biopsy needle ( atec , hologic , bedford , ma ) . the chi - square test was used for discrete data and the t - test for continuous data . statistically significant difference was considered at p < 0.05 . the statistical analysis was performed using medcalc statistical software , version 12.3.0 - 1993 - 2012 medcalc software bvba - medcalc software , broekstraat 52 , 9030 mariakerke , belgium . a systematic review of the breast imaging center 's database was performed to identify all asymptomatic women who were reported to have heterogeneously dense [ figure 1a and b ] or extremely dense [ figure 2a and b ] breast tissue , as defined by the bi - rads atlas , on screening bilateral mammogram performed from july 1 , 2010 through june 30 , 2012 and who received a final assessment birads category 1 , negative or birads category 2 , benign . ( a ) bilateral mammogram cranio caudal view - heterogeneously dense breasts ( b ) bilateral mammogram medio lateral oblique view - heterogeneously dense breasts ( a ) bilateral mammogram cranio caudal view - extremely dense breasts ( b ) bilateral mammogram medio lateral oblique view - extremely dense breasts at the time of the study , the breast imaging center was initiating a policy by which a paragraph was added to the radiologist 's mammogram report to the referring physician of women meeting the above criteria , stating given the dense breast tissue , which may lower the sensitivity of mammography , supplemental screening with breast ultrasound is offered . at our facility , women at high risk for breast cancer ( 2025% or greater lifetime risk of breast cancer ) , as defined by the american cancer society , are recommended to undergo supplemental screening with breast magnetic resonance imaging ( mri ) , and thus were not included in this study . further , not included were women with a personal history of breast cancer , who at our institution undergo diagnostic , not screening mammography , for life . the following data was retrieved for all women included in the study : age , race , family history of breast cancer , personal history of breast biopsy , personal history of biopsy - proven high risk lesion of the breast , age at menarche , age at menopause , parity , age of first pregnancy , and use of hormones ( hormonal contraceptives and hormone replacement therapy ) . in addition , for women who received the offered breast ultrasound , the following data was also retrieved based on the bi - rads atlas : final assessment bi - rads category of the breast ultrasound exam ; if biopsy was recommended , descriptors of ultrasound findings ( solid vs cystic , measurement of longest axis in cm , shape , margins , echogenicity , posterior acoustic features , orientation ) ; and pathology results if biopsy was performed . further , if the final assessment category of the initial screening breast ultrasound was bi - rads 3 , the results of the first two consecutive short - term follow - up ultrasounds were recorded . an interval follow - up increase in size of a mass by more than 20% in the longest axis was considered to warrant biopsy . every screening bilateral mammogram was obtained as 2d digital study on a selenia- hologic unit and was performed by one of six mammography technologists , all of whom are certified in mammography ( registered by the american registry of radiologic technologists ) , with an experience of 17 , 11 , 6 , 6 , 5 , and 4 years , and was interpreted by one of the four board certified breast imagers , with 30 , 11 , 3 , and 2 years of experience in breast imaging . every supplemental bilateral breast ultrasound examination was obtained less than six months from the screening mammogram and was obtained on a dedicated breast ultrasound unit ( ge logic e9 ) with a high - resolution linear - array transducer ( 615 mhz ) by one of the six technologists , two of whom are rdms certified ( registered diagnostic medical sonographer ) and one of whom is arrt certified ( american registry of radiologic technologists ) . two of them with experience in breast sonography for 10 years each , one with 6 years , two with 5 years of experience each , and one with 1-year experience . the breast ultrasound was performed in a standardized hand - held manner with overlapping scans in the radial and antiradial planes , extending from the nipple to the posterior breast tissue . if no abnormal findings were identified , images were documented in the 12- , 3- , 6- , and 9-oclock positions , as well as in the retroareolar region . if any finding was present , images of each finding were obtained and measured in three dimensions . immediately after the breast ultrasound exam by the technologist , the exam was repeated by one of the four board - certified dedicated breast imaging radiologists . the breast imager performed a repeat complete ultrasound scan of the breasts in real time , regardless of whether or not the technologist identified any abnormality and was not blinded to the results of the screening mammogram and the patient 's history and was able to review the breast ultrasound images obtained by the technologist . all ultrasound - guided breast biopsies were performed either with a 14-gauge automated core biopsy needle ( achieve , cardinal health , dublin , ohio ) , or a 9 or 12-gauge vacuum - assisted core biopsy needle ( atec , hologic , bedford , ma ) . the chi - square test was used for discrete data and the t - test for continuous data . the statistical analysis was performed using medcalc statistical software , version 12.3.0 - 1993 - 2012 medcalc software bvba - medcalc software , broekstraat 52 , 9030 mariakerke , belgium . during the study period , a total of 2469 asymptomatic women not at high risk of breast cancer and without a personal history of breast cancer and who received a final assessment bi - rads category 1 , negative , or bi - rads category 2 , benign , at screening bilateral mammogram were evaluated . of these , 1210 ( 49% ) were found to have heterogeneously dense or extremely dense breast tissue . of these , 394 ( 32.5% ) women underwent the offered supplemental screening bilateral breast ultrasound . at the initial round of supplemental screening breast ultrasound , 323 women ( 81.9% ) received a final assessment bi - rads category 1 , negative [ figure 3 ] , or bi - rads category 2 , benign [ figure 4a and b ] and were recommended to undergo routine yearly screening ; whereas 50 women ( 12.9% ) received a final assessment bi - rads category 3 , probably benign [ figure 5a c ] and were recommended to undergo short - term follow - up with breast ultrasound in 6 months [ table 1 ] . two of the women who received a bi - rads category 3 requested biopsy , which was performed . a bi - rads category 4 , suspicious [ figures 6a c and 7a and b ] , with recommendation for biopsy was assigned to 19 women ( 4.8% ) [ table 1 ] . a total of 21 women ( 5.3% ) underwent an ultrasound - guided procedure as a result of the initial round of supplemental screening bilateral breast ultrasound . negative ( a ) bilateral screening breast ultrasound showing a simple cyst in a patient with breast implants - category birads 2 . benign ( b ) bilateral screening breast ultrasound showing a simple cyst with color doppler in the same patient with breast implants- category birads 2 . benign ( a ) bilateral screening breast ultrasound showing a benign appearing mass , likely a fibroadenoma for which a six month follow up ultrasound was recommended- category birads 3 . ( b ) bilateral screening breast ultrasound showing a benign appearing mass , likely a fibroadenoma for which a six month follow up ultrasound was recommended- category birads 3 . probably benign ( c ) bilateral screening breast ultrasound showing a benign appearing mass , likely a fibroadenoma with color doppler for which a six month follow up ultrasound was recommended- category birads 3 . probably benign bi - rads category at initial round of supplemental screening breast ultrasound and at recommended subsequent short - term follow - ups ( a ) bilateral screening breast ultrasound showing a round , heterogenous mass with mixed solid and cystic components and irregular margins . suspicious ( b ) bilateral screening breast ultrasound showing a round , heterogenous mass with mixed solid and cystic components and irregular margins . radial plane . suspicious ( c ) bilateral screening breast ultrasound showing a round , heterogenous mass with mixed solid and cystic components and irregular margins . suspicious ( a ) bilateral screening breast ultrasound showing a palpable mass in the right breast 9:00 axis . suspicious ( b ) bilateral screening breast ultrasound showing a palpable mass in the right breast 9:00 axis with color doppler . suspicious as a result of the first two consecutive short - term follow - ups with breast ultrasound recommended to patients who were assigned a bi - rads category 3 , five more biopsies were recommended and performed ; four biopsy recommendations were generated at the first short - term follow - up cycle and one at the second short - term follow - up cycle [ table 1 ] . overall , a total of 26 women ( 6.6% ) were recommended a biopsy , which was performed in all of them . the most common ultrasound finding for which biopsy was recommended and performed was a solid mass ( 88.5% ) with an average size of 0.9 cm ( range : 0.51.7 cm ) [ table 2 ] . fine needle aspiration of a complicated cyst in two patients ( 11.5% ) was performed and resulted in complete resolution of the cyst with nonsuspicious fluid [ table 2 ] . our study includes only asymptomatic women with dense breast tissue who are not at high risk for breast cancer , which represent the majority of women with dense breast tissue who undergo screening mammography for breast cancer . some prior studies on supplemental screening with breast ultrasound for dense breast tissue have only included women at a high risk for breast cancer , whereas others have included symptomatic women and unilateral breast ultrasound obtained in women with known mammographic abnormalities in the contralateral breast or even in a different quadrant of the ipsilateral breast . moreover , it has been pointed out that there may be methodological flaws in the numerous studies which have previously suggested a link between breast density and the risk of breast cancer . this may be in part because of the problem of trying to extract 3d information from 2d images as stated by kopans . prior studies on this topic have differences in the qualification of the ultrasound exam performers , with some performed only by the radiologist , some only by the technologist , and some by both . performing the supplemental hand - held screening breast ultrasound consecutively by two performers , first by a technologist and then by the interpreting radiologist , as in our study , likely serves to elucidate most mammographically occult findings ; however , it is time consuming and represents a burden on the already limited healthcare resources in a busy clinical practice . an important methodological similarity between our study and some of the prior studies is using the biopsy results and the results of a 1-year follow - up as a reference standard to assess for false negative results , including the occurrence of interval cancers . participants in our study were slightly younger ( mean age of 47.3 years ) as compared to those in other studies with a mean age of participants ranging from 51.2 to 55.2 years . our results have several similarities to the results of previous studies , including the proportion of subjects with bi - rads categories 1 , 2 , and 3 , the biopsy rate , and that fibroadenoma and stromal fibrosis accounted for most pathology findings . moreover , in concurrence with prior studies was the small number of the ultrasound findings that required further evaluation with biopsy , which ultimately is in concordance with the expected small number of the mammographically occult abnormalities that were found with supplemental breast ultrasound . limitations of our study include a small population size , which is likely responsible for the fact that no carcinoma was found . there is only one published study in which the population size is smaller than ours and in which additional breast carcinoma was found with supplemental ultrasound . however , unlike our study , that study included participants with a personal history of breast cancer . in conclusion , our results confirm some of the reported disadvantages of performing supplemental screening with breast ultrasound , in particular the high false - positive rate and a relatively high rate of short interval follow - up , and support the expressed opinions that caution should be exercised when recommending supplemental screening with hand - held bilateral breast ultrasound for all asymptomatic women with dense breast tissue without taking into account other risk factors , the expected large number of women who would undergo this additional test , and the added costs to the health care system .
this report concerns two brothers aged 10 and 18 years with long - standing dysphagia that started at age three and six years respectively . they had been diagnosed as achalasia and treated accordingly . the appearance of additional symptoms and clinical signs required further investigations including abdominal sonography , esophago - gastroduodenoscopy , barium swallow , esophageal manometry , computerized tomography ( ct ) of abdomen and brain , biochemical profiles , and neurologic and ophthalmic evaluations . the results of these extensive investigations along with the clinical evaluations were consistent with allgrove 's syndrome.glucocorticoid therapy was initiated . the management consisted of pneumatic cardiac dilatation and initiation of cortisone treatment . the patients response was impressive and they resumed most of their usual activities .	allgrove 's or triple a syndrome , which was later on named 4a syndrome is a rare autosomal recessive condition characterized by alacrima , achalasia , autonomous neuropathy and acth insensitivity among other features . the syndrome usually presents during the first decade of life with dysphagia , while other signs may be delayed until adulthood . allgrove and his colleagues first described this syndrome in 1978 in two unrelated pairs of siblings ( aged 4 and 6 years).1 all four had achalasia and adrenocortico- tropic hormone ( acth ) insensitivity , three suffered from impaired lacrimation and one had autonomic dysfunction . the syndrome may manifest itself during the first decade of life , with severe hypoglycemia or hypotensive attacks , which may lead to sudden death . allgrove 's syndrome may be an underdiagnosed multi - system disorder in which achalasia and alacrima are the most valuable clinical signs to reach the diagnosis . catastrophic complications can be prevented with adequate cortisol and specific measures such as cardiac pneumatic dilatation or myotomy along with other supportive management . an 18-year - old boy was brought to our emergency room with deterioration of general health , cachexia and weight loss . he was bedridden , had stool and urine incontinence and complained of progressive difficulty of swallowing for some months . he is the first child of healthy non - consanguineous parents and product of full - term delivery . later on , the family noticed the progressive nasal speech and anorexia and lack of weight gain with frequent regurgitation . the patient was subjected repeatedly to esophageal cardiac dilatations . due to the poor response and general health deterioration , after three years of follow - up , for some social reasons , his father stopped taking him to that hospital and started taking him to different hospitals . the father was appreciative of his son 's tolerance by saying that despite all the problems , he had not seen any tears shed by his son . apart from his younger brother who had a milder form of similar symptoms , the other siblings , parents and relatives were all free of symptoms . he was pale with hyper - pigmentation of skin , gums , and palmar creases . his face was long and thin with narrowed upper lips down - turned mouth and microcephaly . the joint motions were limited because of muscle contractures , spastic tetraparesis with mild ataxia , deep tendon reflexes were increased in all four limbs . his basic investigations showed moderate anemia of hemoglobin ( hb ) of 10 g / dl . cortisol level at 8 am was less than 1.1 ug / dl and acth was 54.1 . barium swallow study and esophageal manometry features were consistent with achalasia . he was seen and evaluated by an ophthalmologist and schirmer 's test was needed . this revealed dry eyes i.e. alacrima while , ct brain and orbit tomography showed reduced lacrimal gland tissue . patient during admission in the hospital his management included full supportive therapy that included hydration , nutrition , physiotherapy and artificial tears . the achalasia was managed by two sessions of pneumatic dilatations and he was kept on 10 mg prednisone / day as a maintenance dose . during one year of follow - up , he exhibited significant improvement and was able to eat well , walk without any support and he gained 25 kg weight i.e. , his body mass index ( bmi ) increased from 13.3 to 21.3 ( figure 2 ) . his main presentation was frequent regurgitation , weight loss and failure to thrive . at the age of three , his physical examination revealed an under - developed child with nasal speech . at the age of four , the diagnosis of achalasia was established and he was subjected to heller 's surgery . he was found to be thin , underweight and microcephalic . the following investigations were performed in our hospital : barium swallow , esophageal manometry which indicated achalasia and upper gi endoscopy which showed good functioning myotomy . based on the clinical appearance , achalasia as well as alacrima , an early and slow progressing allgrove 's syndrome was diagnosed . allgrove 's syndrome is considered an autosomal recessive disorder with variable presentations.12 recent studies have identified mutation in the aaa syndrome of a candidate gene on chromosome 12q13 in such patients.3 prpic et al ( 2003 ) demonstrated the marked phenotypic variability in three patients with genetically confirmed triple a syndrome . two patients had achalasia , alacrima and adrenocortical deficiency as well as neurologic and autonomic dysfunction . all patients were homozygous for mutations in the triple a syndrome gene.3 the age of onset of symptoms is variable , the syndrome usually presenting during the first decade of life with dysphagia or severe ( occasionally fatal ) hypoglycemic or hypotensive attacks related to adrenocortical insufficiency.45 in our two cases , achalasia was the initial alarming symptom causing vomiting , dysphagia , recurrent chest infection and failure to thrive , which required frequent hospital admissions . these symptoms were noticed at the age of three and six respectively , which helped in reaching the diagnosis of achalasia . the association of dry eyes together with achalasia was an important clinical sign in support of allgrove 's syndrome . both patients had proven alacrima , which impressed the parents as high pain threshold and tolerability . the biopsy obtained from his gland may show neuronal degeneration and depletion of secretory granules in the acinar cell.56 conjunctival congestion and irritation should be the only sign of alacrima which could be confirmed by the schirmer 's test . the younger one had no debilitating finding and had a normal life and good school performance . the index case had areas of hyper - pigmentation , most obvious over the buccal mucosa and gums . peripheral motor and sensory neuropathy reveal muscle wasting , hyperreflexia , dysarthia , nasal speech , ataxia and autonomic dysfunction.68 all these signs were present in the first case contributing significantly to his morbidity.910 adrenocortical function was preserved in case 2 while the index case was remarkably affected with a typical pattern of acth insensitivity . baseline acth , cortisol level and acth stimulation test are used to evaluate adrenal insufficiency . glucocorticoid replacement therapy seems to have no influence on the development and progression of neurological features . no explanation for the association of achalasia , alacrima and adrenal unresponsiveness to acth in the triple a syndrome is available . it has been thought that the acth receptor gene would provide the link to explain the association of the three main features of the syndrome , since there is evidence that acth has some neuropathic effects.1112 imaging studies such as ultrasonography , computerized tomography and magnetic resonance might be informative if neurological abnormalities are noticed . contrast swallow study , esophagoscopy and manometry give the definite diagnosis of achalasia and should be considered among the preliminary investigations for the diagnosis . in both patients , the index case responded to graded pneumatic dilatation and his younger brother had open cardiomyotomy at age four ( six years prior to the diagnosis of allgrove 's syndrome ) . careful replacement of glucocorticoids in patients with adrenal insufficiency and management of achalasia either via endoscopic or surgical approach are cornerstones of the treatment . in long term follow - up of adults , increased risk of gastroesophageal reflux ( ger ) and its epitheloid changes have been reported.13 myotomy with antireflux procedure will , therefore , be a better choice and in case of pneumatic dilatation , a small dose of proton pump inhibitors ( ppis ) might be needed . additionally , the usage of artificial tears and application of topical lubricants with other supportive therapy can improve the general outcome . he is able to perform his daily routines without any support , communicates well and has gained 25 kg . allgrove 's syndrome may be an underdiagnosed disorder . a high index of suspicion is required when patients present with such complex symptoms as failure to thrive ( dysphagia ) , crying without tears ( alacrima ) , nasal speech and seizures ( hypoglycemia ) . diagnosis can be confirmed by esophageal manometry , ophthalmic assessment , biochemical study and neurological evaluation .
a well - proportioned face combines features that are balanced and symmetrical . any structural alteration that leads to facial asymmetry causes esthetical and psychological disturbances . lipoatrophy is one such condition , which results in loss of subcutaneous fat layer and manifests as a depression . although many subtypes with varying clinical and etiological backgrounds exist , the idiopathic form is rare and facial involvement is the rarest . computed tomography is one of the accepted diagnostic tools to determine the atrophic layer of facial anatomy . this report presents the clinical types , diagnosis , and management of a case of facial lipoatrophy .	lipodystrophy broadly refers to a disturbance in the production , utilization , and storage of fat . lipoatrophy is a sharply defined disappearance of the subcutaneous fat , without exudative reactions or appreciable fibrosis ( marble and smith 1942 ) . lipoatrophy is divided into generalized , partial ( extensive , but not generalized ) , and localized ( limited to a localized area ) types . there is no universal or precise proven reason for lipoatrophy ; however , literature suggests impairment of adipocyte differentiation , adipocyte apoptosis , and mitochondrial dysfunction , as the heterogeneous pathogenesis , reflecting the different subtypes . the congenital type is a genetic disturbance , presenting with a generalized absence of fat within the first year of life and is followed by a series of abnormalities like insulin resistance and acanthosis nigricans before adolescence . the onset is during childhood , with widespread panniculitis , and a strange physical stature . it has a characteristic beginning of progressive subcutaneous fat loss in the face and scalp , and then spreads up to the iliac crests . children between five and eight years are the target group , with girls outnumbering boys by 4:1 . iatrogenic causes of lipoatrophy include complications of injected medications like insulin , corticosteroids , antibiotics ( penicillin g ) , iron , heparin , and vaccines . hiv - positive patients on anti - retroviral therapy , especially , nucleoside reverse transcriptase inhibitors ( nrts ) and protease inhibitors ( pi ) manifest lipoatrophy as an adverse drug effect . a 28-year - old female patient reported to our institution with a complaint of a depression appearing on the right lower facial region . it had started as a small depression two years earlier , with no accompanying pain or discomfort , which progressed to the present state of asymmetry and abnormal appearance . her medical records showed no history of facial trauma or dental infections . on examination , the facial asymmetry was found to be due to deficiency of the fat layer in the right parasymphyseal region , extending inferiorly to the lower border of the mandible [ figure 1a ] . localized idiopathic subcutaneous atrophy of the face was considered as a clinical diagnosis and the relevant diagnostic workup was started . right profile view of the patient shows the atrophic region with pigmentation . an en - face photograph and profile views of the patient were taken and analyzed together , to assess the degree of asymmetry [ figures 1a and b ] . ultrasonography using , 7 - 10 mhz frequency linear probe ( volvuson 730 pro expert ge machine ) revealed the normal superficial skin layer bilaterally . the thickness of subcutaneous plane was 0.22 cm on the right side and 0.28 cm on the left side . altered echogenicity was noted on the right masseter muscle . on the affected side , the muscle thickness was 0.83 cm , whereas , on the normal side , it was 1.26 cm [ figures 1c and d ] ultrasound image reveals normal facial anatomy on the left side and altered echogenicity on the right cheek at the subcutaneous and massetric layer . ultrasound image of the affected side , allows measurement of muscle thickness in both open and closed mouth positions . the serial axial and coronal ct sections of the craniofacial bones obtained with multiplanar and volume - rendered reconstructions confirmed the asymmetry of the subcutaneous soft tissues , which were atrophied on the right cheek region . the underlying muscles and bones appeared normal [ figures 1e and f ] coronal ct shows subcutaneous atrophy of fat on the right side . axial ct demonstrates subcutaneous atrophy at the level of the right mandible . patient 's face after soft tissue reconstruction . clinical investigations showed normal values for complete blood count , blood glucose , serum cholesterol , and triglycerides . the antinuclear antibody ( ana ) test was negative . based on the clinical and imaging , we concluded the final diagnosis as localized , idiopathic involutional grade 1 lipoatrophy of the face . autologous fat harvested from the abdomen was injected to the subcutaneous plane to rebuild the sunken area [ figures 1 g and h ] the subcutaneous fat provides volume and mobility , and is supported by fibrous retinacular cutis , which connects the dermis to the muscle apponeurotic system . the subcutaneous layer is critical , because , wasting of subcutaneous fat results in atrophy , whereas , its increased thickness leads to lengthening of the retinacular fibers , therefore , weakness and distention . paucity or complete absence of fat in the confined subdermal layer , with lack of inflammatory signs , is known as idiopathic localized involutional lipoatrophy ( ilil ) . peters and winkelmann first reported this condition , as a sharply demarcated skin depression without any epidermal alteration . the features of facial lipoatrophy include sunken cheeks , deep folds in the nasolabial region , skin depression at the temples , sides of forehead , and around the eye sockets . spontaneous regression in few weeks is usual , however , in some cases the lesion may persist and cause cosmetic concerns . grade 1 mild flattening or shadowing on one or more facial regions ; no prominent bony land marks and no visibility of the underlying musculature . grade 3 moderate concavity of one or more facial regions , prominence of bony land marks , and possible visibility of the underlying musculature . grade 5 severe depression of one or more facial regions , severe prominence of bony land marks , and clear visibility of the underlying musculature . computed tomography ( ct ) plays an useful role in the diagnosis of focal lipoatrophy as fat can be easily appreciated on ct . us can demonstrate asymmetry of the face and loss of fat in focal lipoatrophy but it has less sensitivity than ct . to improve esthetics , common treatment modalities , with evidence of safety and efficacy the possibility of severe infection , contour abnormalities , and facial nerve and muscle damage , restrict the use of permanent type of fillers like silicone oil . non - permanent fillers , being biodegradable , can diminish over time and may necessitate a revamp , but they do not cause foreign body reactions or granulomas . injection of a biodegradable and bioabsorbable poly - l - lactic acid ( pla ) filler causes cutaneous thickening by fibrous connective tissue formation over a month . the call for multiple treatment sessions , cost , and transitory relief , limit its use for augmentation . autologous fat transfer ( aft ) is a secure and cost - effective option , exclusively for non - hiv patients with lipoatrophy . in our patient , as the abnormality was a mild cosmetic disorder , with no disability , needle extraction of adipose tissue from the abdomen and injecting this at the site of the atrophy was done with the aim of ensuring rapid revascularization and less resorption . a soft , slightly curved , and well - defined anatomy with esthetic satisfaction was achieved . frequent patient follow - up is essential to monitor the acceptance and consequences , and to see if there is a need for repeating the treatment . facial lipoatrophy in otherwise healthy individuals is uncommon and patients usually present for cosmetic reasons .
scleroderma ( ssc ) renal crisis has been reported to be associated with anti - rna polymerase i and iii ( rnap i / iii ) antibodies in caucasians and the japanese . however , no report is available for korean ssc patients . here , we describe the case of a 65-yr - old female ssc patient who developed renal crisis and whose serum contained anti - rnap i / iii antibodies . she was finally diagnosed as having diffuse cutaneous ssc based on skin thickening proximal to the elbows and knees . sudden hypertension , oliguria , and pulmonary edema were features of her renal crisis . despite the use of captopril and adequate blood pressure control , her renal function deteriorated . subsequent renal biopsy findings showed severe fibrinoid necrosis with luminal obliteration in interlobar arteries and arterioles consistent with ssc renal crisis . serum anti - rnap i / iii antibodies were detected by radioimmunoprecipitation . this is the first report of a renal crisis in a korean ssc patient with rnap i / iii antibodies .	systemic sclerosis ( ssc , scleroderma ) is an autoimmune disease characterized by the accelerated accumulation of extracellular matrix and by various immunologic abnormalities ( 1 ) . serum autoantibodies are helpful markers because they have been correlated with certain clinical features of ssc ( 2 , 3 ) . of the various ssc - related antibodies , anti - rna polymerase ( rnap ) antibodies are known to be ssc specific and to be present in 4 - 33% of ssc patients ( 4 - 8 ) . there are three classes of rnaps ( rnaps i , ii , and iii ) ( 9 ) and anti - rnap i and iii ( i / iii ) antibodies have been detected exclusively in ssc patients ; moreover , the presence of these antibodies is known to be associated with diffuse cutaneous involvement and renal crisis ( 5 , 6 , 10 , 11 ) . although the association between scleroderma renal crisis and anti - rnap i / iii antibodies has been reported in caucasians and in the japanese , no such report has been issued in the korean ssc population . furthermore , autoantibodies in korean ssc patients show several distinctive features ; 1 ) no association between disease subset and autoantibodies , such as , anti - topoisomerase i ( anti - topo i ) or anticentromere antibody ( aca ) , 2 ) a much lower prevalence of aca in a limited subset ( 6.7 - 8.0% vs. 44% in caucasians and 37% in the japanese ) , and 3 ) no significant difference in the clinical characteristics of disease subsets , except for more frequent musculoskeletal involvement in a limited subset ( 12 - 14 ) . here , we report for the first time a case of renal crisis in a korean ssc patient with serum anti - rnap i / iii antibodies detected by radioimmunoprecipitation ( 5 ) . a 65-yr - old female visited the rheumatology clinic due to the thickening of hand and facial skin with digital pallor and cyanosis on cold exposure , which had developed 2 months previously . on the first visit to the clinic , her blood pressure was 130/80 mmhg and physical examination revealed skin thickening on the fingers of both hands , and on the right hand dorsum and right forearm . she was diagnosed as having systemic sclerosis of the limited cutaneous subset based on the american rheumatism association preliminary criteria ( 15 ) . laboratory data showed white blood cells at 8.3910/l , hemoglobin 12.1 g / dl , platelets 24010/l , esr 8 mm / hr ( normal range : 0 - 20 ) , got 21u / l , gpt 18 u / l , total bilirubin 0.6 mg / dl , albumin 3.7 g / dl , bun 11 mg / dl , and creatinine 0.7 mg / dl . pulmonary function testing produced the following ; forced vital capacity , 73% ; forced expiratory volume in 1 sec , 79% ; and diffusing capacity of carbon monoxide over volume of alveoli , 121% . high resolution computed tomography of the lungs revealed old tuberculosis in the right lower lobe with pleural thickening and calcifications without evidence of interstitial lung disease . prazosin 1 mg / day was administered for raynaud 's phenomenon and intermittent antihistamines for skin pruritus . twenty - two months after the diagnosis of limited ssc , her skin thickening began to rapidly progress above elbows and knees , and finally involved the trunk . the diagnosis was converted to ssc of the diffuse cutaneous subset and she was started on d - penicillamine 250 mg / day . a month later , she visited the emergency room due to the sudden onset of facial edema and severe dyspnea . her blood pressure was 220/134 mmhg , heart rate 120 beats / min , respiration rate 48/min , and body temperature 35. she reported that the dyspnea had begun 10 days previously and that her urine output had decreased markedly . a physical examination revealed facial and pre - tibial edema , and pulmonary rales in the whole lung field . laboratory data showed hemoglobin at 9.9 g / dl , ldh 703 iu / l , total bilirubin 2.9 mg / dl , indirect bilirubin 1.8 mg / dl , and schistocytes with polychromasia on peripheral blood smear , suggesting intravascular hemolysis . urinalysis and microscopic examination showed mild albuminuria ( 1+by dipstick test ) , hematuria ( 100 red blood cells per high power field with dysmorphic red blood cells > 90% ) , and pyuria ( 20 - 29 white blood cells per high power field ) . urine culture grew no organisms . azotemia was also detected with a bun of 31 mg / dl and a creatinine of 2.2 mg / dl . her chest radiograph showed cardiomegaly and bilateral hilar infiltrates consistent with pulmonary edema . she was transferred to intensive care unit , intubated , and assisted with a mechanical ventilator . under a diagnosis of scleroderma renal crisis , captopril was begun , but failed to reverse a deteriorating renal function despite successful blood pressure control within 2 days . renal biopsy showed severe fibrinoid necrosis with luminal obliteration in interlobar arteries and arterioles , strongly suggesting scleroderma renal crisis ( fig . immunofluorescence staining showed no evidence of immune complex or autoantibody deposition . because scleroderma renal crisis has been reported to be associated with anti - rnap i or iii antibodies , we tested whether this patient had anti - rnap antibodies in her serum . indeed , anti - rnap i / iii antibodies were detected by radioimmunoprecipitation using 35s - methionine - labeled k 562 cells ( fig . this is the first case report of scleroderma renal crisis with the presence of rnap i / iii antibodies in the korean population . renal crisis in this patient was diagnosed by accelerated hypertension and acute renal failure with a typical renal pathology . the clinical findings in association with the presence of anti - rnap i / iii antibodies in this case are consistent with previous reports of ssc patients ( 5 , 6 , 10 , 11 , 16 , 17 ) , with respect to ; 1 ) renal crisis in association with anti - rnap i / iii antibodies , 2 ) the presence of anti - rnap i / iii antibodies in a diffuse subset patient , and 3 ) the absence of anti - topo i in the presence of anti - rnap i / iii antibodies . although not all ssc patients with anti - rnap i / iii antibodies develop renal crisis , renal crisis occurs in anti - rnap i / iii antibody positive patients much more frequently than in negative patients ( 5 , 6 , 11 ) . moreover , it is well known that the early administration of angiotensin converting enzyme ( ace ) inhibitors can improve renal outcome . therefore , knowing whether anti - rnap i / iii antibodies are present before renal crisis onset should alert physicians to the possibility of renal crisis and facilitate the initiation of prompt treatment . according to steen et al . ( 18 ) , 44% ( 24/55 ) of scleroderma renal crisis patients who received ace inhibitors , either died or required permanent dialysis while 56% ( 31/55 ) of them did not require dialysis at all or needed only transient dialysis . the factors that were associated with poor renal outcome were old age ( > 55 yr ) ( odds ratio , 12.28 ; p=0.002 ) and the presence of congestive heart failure ( odds ratio , 3.89 ; p=0.03 ) . this patient showed both factors and administration of captopril failed to improve her renal function . the poor response to ace inhibitor in this patient suggests that her renal function had irreversibly deteriorated prior to initiation of medical treatment because she had symptoms of acute renal failure for at least 10 days before she was treated . rnaps ( rnaps i , ii , and iii ) are multimeric proteins that are composed of 8 - 14 subunits of 10 to 220 kda ( 9 ) . the two largest subunits are unique to each class and are readily distinguishable by their characteristic mobilities on sds - polyacrylamide gel ( 6 , 7 ) . because of the size and complexity of rnaps , it has proven difficult to develop methods for detecting anti - rnap antibodies that are applicable in a clinical laboratory setting despite the potential benefit of knowledge on the presence of anti - rnap i / iii antibodies . however , recently , it was reported that anti - rnap antibodies can be detected reliably using an enzyme - linked immunosorbent assay method ( 17 , 19 ) , which might help measure these antibodies routinely in the near future . in summary , we report a case of renal crisis in the presence of anti - rnap i / iii antibodies in a korean ssc patient of a diffuse cutaneous subset . further studies with a sufficient number of patients are needed to confirm the association between the presence of anti - rnap i / iii antibodies and clinical features in the korean population .
objectivewe evaluated the feasibility of angiographic computed tomography ( act ) for visualizing stent material in patients who underwent intracranial or extracranial stent placement to treat atherosclerotic lesions or stent assisted coil embolization.materials and methodswe performed intrarterial and intravenous act on biplane angiography system equipped with flat panel detectors ( axiom arits dba ; siemens medical solutions , forchheim , germany ) . vistipaque 320 was injected for contrast medium , total 150 ml at flow rate of 5 ml / s through artery and 77 ml at flow rate of 3.5 ml / s through vein.resultsact is a new imaging modality that provides a clear visualization of stent strut.conclusiontherefore this new application has potential to become the noninvasive option for follow - up after endovascular surgery using stents .	angiographic computed tomography ( act ) is a new technique that provides a highest spatial resolution and high contrast resolution derived from a rotational acquisition of a c - arm - mounted flat - panel detector.4 ) we demonstrated the feasibility and diagnostic value of intrarterial ( ia ) and intravenous ( iv ) -act for pre or postprocedure imaging for stent angioplasty and stent assisted coil embolization of cerebral aneurysm . conventional digital subtraction angiography ( dsa ) and act were performed on biplane angiography system equipped with flat panel detectors ( axiom arits dba ; siemens medical solutions , forchheim , germany ) . the parameters for acquisition of rotational datasets ( 20s-1k protocol ) : 20 seconds rotation , 538 projections , 220 total angle , no zoom ( detector size 30 40 cm ) , ctdiw approximately 22 mgy ( manufacturer 's information ) . post processing of the image data to a volume dataset was performed on a leonardo workstation ( dynact , inspace 3d software , siemens , erlangen , germany ) . the software includes the application of system - specific filter algorithms in order to correct for beam hardening , radiation scatter , truncated projections and ring artifacts . post processing resulted in volume datasets each defined by a batch of about 400 slices in a 512 512 matrix . the contrast medium used in the ia - act examination was visipaque320 ( ge healthcare , carrigtohill , ireland ) the contrast medium was injected to a volume of 150 ml ( contrast 100 ml with saline 50 ml mixed ) at a flow rate of 5 ml / s , and the start delay for rotational acquisition was 20 seconds(intracranial ) or 15 seconds(vao , cc - ic ) . the contrast medium was injected for iv - act to a volume of 77 ml into a artery at a flow rate of 3.5ml / s , and the start delay for rotational acquisition was 2 seconds . conventional digital subtraction angiography ( dsa ) and act were performed on biplane angiography system equipped with flat panel detectors ( axiom arits dba ; siemens medical solutions , forchheim , germany ) . the parameters for acquisition of rotational datasets ( 20s-1k protocol ) : 20 seconds rotation , 538 projections , 220 total angle , no zoom ( detector size 30 40 cm ) , ctdiw approximately 22 mgy ( manufacturer 's information ) . post processing of the image data to a volume dataset was performed on a leonardo workstation ( dynact , inspace 3d software , siemens , erlangen , germany ) . the software includes the application of system - specific filter algorithms in order to correct for beam hardening , radiation scatter , truncated projections and ring artifacts . post processing resulted in volume datasets each defined by a batch of about 400 slices in a 512 512 matrix . the contrast medium used in the ia - act examination was visipaque320 ( ge healthcare , carrigtohill , ireland ) the contrast medium was injected to a volume of 150 ml ( contrast 100 ml with saline 50 ml mixed ) at a flow rate of 5 ml / s , and the start delay for rotational acquisition was 20 seconds(intracranial ) or 15 seconds(vao , cc - ic ) . the contrast medium was injected for iv - act to a volume of 77 ml into a artery at a flow rate of 3.5ml / s , and the start delay for rotational acquisition was 2 seconds . a 53-year - old female was treated because of symptomatic 85% stenosis at the cervical internal carotid artery ( ica ) . after a protective device ( angiogurd rx ; cordis ) was placed in the distal cervical segment of the ica , 8 40 mm device ( precise ; cordis ) stent was applicated at the lesion . because significant residual stenosis was present immediately after stenting , post - dilation of the stent was performed by using a 6 30 mm balloon . act showed incomplete stent deploying because extensive calcification in the carotid wall was compressing the segment of the stent . the extent of calcification in the circumference of the arterial wall was more clearly revealed in axial view . this extensive wall calcification explained why the stent could not be fully deployed over its entire length ( fig . 1 ) . a 66-year - old man presented with recurrent left hemiparesis . diffusion magnetic resonance imaging ( mri ) of the brain showed a minimal acute infarction in the basal ganglia ( bg ) and periventricular white matter ( pvwm ) on the left . cerebral angiography showed severe stenosis ( 85% ) of the m1 segment of the right middle cerebral artery ( mca ) . placement of a 2.5 8 mm balloon - expandable stent ( endeavor ; medtronic scientific , roncadelle ( bs ) , italy ) into the right m1 segment was successfully performed , with completely reestablishing the lumen and restoring normal flow . although the stent is not completely identified on dsa and is only slightly more visible on nonsubtracted views , act can detect the stent completely in every plane . the axial view demonstrated full and uniform deployment of the stent over its entire length . three dimensional ( 3d ) dsa was performed and wide neck based aneurysm was confirmed . initially self expandable stent 4.5 20 mm ( neuroform stent ; ~~~ ) was placed to entirely cover neuroform neck . after stenting , ia - act was performed just before coil embolization in order to ensure perfect stent positioning . total of 5 platinum microcoil were inserted in the paraclinoid aneurysm , which completely occluded the lesion . act performed after coil embolization allows to define the exclusion of stent strut movement or coil protrusion with minimal beam - hardening artifacts ( fig . a 53-year - old female was treated because of symptomatic 85% stenosis at the cervical internal carotid artery ( ica ) . after a protective device ( angiogurd rx ; cordis ) was placed in the distal cervical segment of the ica , 8 40 mm device ( precise ; cordis ) stent was applicated at the lesion . because significant residual stenosis was present immediately after stenting , post - dilation of the stent was performed by using a 6 30 mm balloon . act showed incomplete stent deploying because extensive calcification in the carotid wall was compressing the segment of the stent . the extent of calcification in the circumference of the arterial wall was more clearly revealed in axial view . this extensive wall calcification explained why the stent could not be fully deployed over its entire length ( fig . diffusion magnetic resonance imaging ( mri ) of the brain showed a minimal acute infarction in the basal ganglia ( bg ) and periventricular white matter ( pvwm ) on the left . cerebral angiography showed severe stenosis ( 85% ) of the m1 segment of the right middle cerebral artery ( mca ) . placement of a 2.5 8 mm balloon - expandable stent ( endeavor ; medtronic scientific , roncadelle ( bs ) , italy ) into the right m1 segment was successfully performed , with completely reestablishing the lumen and restoring normal flow . although the stent is not completely identified on dsa and is only slightly more visible on nonsubtracted views , act can detect the stent completely in every plane . the axial view demonstrated full and uniform deployment of the stent over its entire length . three dimensional ( 3d ) dsa was performed and wide neck based aneurysm was confirmed . initially self expandable stent 4.5 20 mm ( neuroform stent ; ~~~ ) was placed to entirely cover neuroform neck . after stenting , ia - act was performed just before coil embolization in order to ensure perfect stent positioning . total of 5 platinum microcoil were inserted in the paraclinoid aneurysm , which completely occluded the lesion . act performed after coil embolization allows to define the exclusion of stent strut movement or coil protrusion with minimal beam - hardening artifacts ( fig . however , introduction of small , low profile , high flexibility and excellent tractability stents makes available for wide use of stent for intracranial , extracranial atherosclerotic disease and stent assisted aneurysm coiling , resulting restenosis rates ranging from 8% to 30%.8)11 ) also complications of stent assisted coil embolization such as incomplete stent deployment , stent migration , coil dislocation , and in - stent stenosis during the procedure or the follow period are still yet to be resolved . therefore , pre- , intra- and post - procedural imaging studies , which can help to visualize the exact position of the stent and to identify significant isr , are very crucial . however , it still carries a 0.5% to 0.8% risk of permanent neurological impairment.5)15 ) in addition , dsa is difficult to be done in outpatient clinic and for patients with poor compliance , and it is also not a good imaging tool for follow - up ( f / u ) examination . consequently , these problems force us to use other evaluation technique , such as brain cta , doppler sonography and plane x - ray . brain cta can be used to assess vascular patency after stents . however , in vitro studies have demonstrated that the degree of isr tends to be overestimated.12 ) duplex sonography also plays an important role in the pre- and post - procedure , especially in carotid stenosis , but the actual accuracy is dependent on the examiner . fluoroscopic and plain radiography can also be used , but these modalities are used only in cases where self expandable stent was applied . this type of stent is visible only at the proximal and the distal end where the radiopaque markers are located . because of these problems of previous evaluation tools , many additional imaging techniques are introduced nowadays , including act . synonyms of act are flat panel angiographic tomography , flat panel detector angiographic ct or system , flat - panel ct , angiographic c - arm tomography , cone beam c - arm system , dyna ct ( siemens ) , xper ct ( philips , amsterdam , the netherlands ) . benefits of act are as follows : 1 ) examinations can be performed on an outpatient basis 2 ) the risk of neurological complications is low 3 ) the examination can be performed quickly , in a time similar to that required for a cta 4 ) the radiation exposure for the patient from a rotational acquisition is lower than that from a cta examination ( manufacturer 's information 22 mgy vs. 50 mgy ) 5 ) the patients do not have to be moved to ct scanner in order to confirm the position of the stent or coil during the procedure or immediately after the procedure.10 ) as shown previously , we demonstrated that ia and iv act can be used as another diagnostic option for stents during procedure and the follow - up period , because it can reliably depict the lumen of small - vessel stents with high spatial resolution.6 ) utilizing act imaging may enable adequate visualization of the stent position and especially the stent struts.2)3 ) all intracranial vessels can be viewed simultaneously in high - quality images , while ct - like images of the brain parenchyma are provided as well . however , limitations arise with artifacts , including movement artifact , and with calibration problems that deteriorate image quality . the patient has to lie nearly motionless for about 20 seconds , requiring a high level of compliance , which is not always feasible in patients suffering from cerebrovascular disease.4 ) another limitation is that the contrast differentiation is inferior in areas of low radiographic contrast compared to conventional ct imaging . also , reconstruction still requires 5 to 10 minutes , and the dynact procedure has not been accepted as a ct scan modality under the insurance system as it is still considered as fluororadiography . meanwhile , stent - assisted aneurysm coiling is well established in clinical routine.1)7)9)13)14 ) however , fluoroscopic and plain radiographic visibility of the stent struts is especially limited by the low profile of these highly flexible devices , with only the proximal and distal radiopaque stent markers visible . this may render it difficult to exactly assess stent position and adaptation of stent struts to the vessel wall.3 ) act uses rotational , c - arm mounted flat panel detector technology , capable of high spatial resolution volumetric imaging.11 ) using this act imaging may enable adequate visualization of stent position and especially the stent struts.2)3 ) even though this new method needs to be evaluated in a larger number of patients , it introduces many advantages for patients and surgerons as a new option in the imaging of endovascular treatment and cerebrovacular disease . utilization of the angiography ct ( dyna ct ) is a promising new technique for minimally invasive follow - up after endovascular stent and may be helpful in complex stent - assisted aneurysm coiling procedures .
vitamin d deficiency is now recognized as a condition of increasing prevalence worldwide . vitamin d has an established role in calcium and bone metabolism ; however , more recently associations with vitamin d deficiency and risk of developing diabetes , diabetes complications , and cardiovascular disease have all been acknowledged . the vitamin d receptor is ubiquitously expressed , and experimental , in vitro , and in vivo studies strongly suggest a role in regulating the transcription of multiple genes beyond calcium homeostasis . these include antiproliferative , immunomodulatory , angiogenic , inhibition of the renin angiotensin aldosterone system , and neurotrophic factor expression . observational studies report a strong association between vitamin d deficiency and cardiovascular and metabolic disorders ; however , there remains a paucity of large long - term randomized clinical trials showing a benefit with treatment . an increasing body of literature suggests a possible pathogenetic role of vitamin d in the long - term complications of diabetes and vitamin d deficiency may also exacerbate symptoms of painful diabetic peripheral neuropathy . it remains unknown if supplementation of vitamin d to normal or non - deficient levels alters pathogenetic processes related to diabetic microvascular complications . with the high prevalence of vitamin d deficiency in patients with diabetes and putative mechanisms linking vitamin d deficiency to diabetic complications , there is a compelling argument for undertaking large well - designed randomized controlled trials of vitamin d supplementation .	there is an epidemic of vitamin d deficiency , with over one billion people worldwide affected . our previously published uk data indicates that vitamin d deficiency ( 25-hydroxyvitamin d [ 25(oh)d ] < 30 ng / ml ) occurs in 91% of patients with diabetes , with severe deficiency [ 25(oh)d < 10 ng / ml ] in 32% of patients . the purpose of this review was to examine the association between vitamin d status and cardiovascular disease , with particular reference to diabetes mellitus and its complications . this review is based on previously conducted studies and does not involve any new studies of human or animal subjects performed by any of the authors . vitamin d is a lipid - soluble , secosteroid hormone which is primarily associated with calcium homeostasis . synthesis of vitamin d occurs predominantly in the skin from dehydrocholesterol , the shared common precursor with cholesterol , and is dependent on sun exposure . ultraviolet b light of wavelengths between 280 and 315 nm is optimal for the conversion of 7-dehydrocholesterol to previtamin d3 , which is then converted to vitamin d3 in the skin . during summer , exposure to midday sunlight for 2030 min , two or three times per week is thought to be sufficient in generating adequate levels of vitamin d for a fair - skinned individual . the amount of synthesized vitamin d is reduced in darker - skinned , older , and obese individuals . other sources of vitamin d include diet ( food that contains natural vitamin d or fortified vitamin d ) and dietary supplements . dietary vitamin d usually contains vitamin d3 ( cholecalciferol ) with few natural sources containing vitamin d2 ( ergocalciferol ) . vitamin d from the skin and diet is modified in the liver to generate 25(oh)d . active vitamin d is formed when further conversion takes place in the kidneys forming 1,25-dihydroxyvitamin d [ 1,25(oh)2d3 ( activated vitamin d ) ] .table 1significant risk factors for vitamin d deficiencyincreasing ageuse of sunscreenpigmented skinhouse - bound patientsobesitynorthern latitudesmedication ( antiepileptic / antiretroviral drugs)renal diseaseliver diseasemalabsorption syndrome significant risk factors for vitamin d deficiency vitamin d status is defined by measuring the level of 25(oh)d in the blood owing to its longer half - life in plasma , where it forms a circulating reservoir of vitamin d nmol / l ) is considered as sufficient , 2030 ng / ml ( 5075 nmol / l ) as insufficient , 1020 ng / ml ( 2550 nmol / l ) as deficient , and < 10 ng / ml ( < 25 nmol / l ) as severely deficient . the us institute of medicine ( iom ) released guidance in 2011 in which 25(oh)d equal to 20 ng / ml was considered to be adequate for 97.5% of the population ; however , this has been heavily criticized by the endocrine society [ 7 , 8 ] and they recommend that 25(oh)d concentration should exceed 30 ng / ml , to maximize the effect of vitamin d on calcium , bone , and muscle metabolism . cutoffs for vitamin d status are detailed in table 2 . both the iom and the endocrine society have arbitrated respective cutoffs for optimal vitamin d status ; however , these are based on bone metabolic data and not on cardiometabolic data . optimal levels of 25(oh)d for possible prevention of cardiometabolic outcomes remain to be elucidated .table 2serum 25-hydroxyvitamin d concentrations and status25(oh ) vitamin d concentration 25(oh ) vitamin d status<10 ng / mlsevere deficiency10<20 ng / mldeficient20<30 ng / mlinsufficient30 ng / mladequate100 ng / mlpossible toxicity multiply by 2.5 to convert to nmol / l serum 25-hydroxyvitamin d concentrations and status multiply by 2.5 to convert to nmol / l vitamin d insufficiency which is not low enough to cause bone disease is significantly associated with cardiovascular disease . there is a ubiquitous expression of vitamin d receptors ( vdr ) in virtually every human tissue , and vitamin d is an important regulator of literally hundreds of genes regulating key biological processes from cell division to apoptosis . vdr is widely distributed in many tissues , which suggests a putative biological response with its activation , implying that vdr has multiple functions beyond calcium homeostasis . once 1,25(oh)2d3 binds to vdr , it interacts with the retinoid x receptor ( rxr ) forming a heterodimer that binds to vitamin d responsive elements in the region of genes directly controlled by 1,25(oh)2d3 . the action of vitamin d in a particular cell depends on the vitamin d - mediated gene activation , transcriptional response , and protein formation . for example , in addition to stimulating intestinal calcium and phosphate absorption to effect skeletal and mineral ion homeostasis , 1,25(oh)2d3/vdr - rxr also regulates the expression of a plethora of genes in non - calcemic tissues . this extensive vitamin d - ome allows the concerted genomic and rapid actions of 1,25(oh)2d3 to possibly have an interplay with such varied diseases as osteoporosis , cancer , diabetes , atherosclerosis , vascular disease / calcification , and infection . furthermore , the vdr gene is expressed within developing neurons of rodent dorsal root ganglia , which may suggest a role for vitamin d in peripheral nervous system development and nociception . subjects with severe vitamin d deficiency ( 25(oh)d < 10 ng / ml ) experienced a hazard ratio of 1.80 [ 95% confidence interval ( ci ) , 1.053.08 ] for developing a first cardiovascular event 5 years after follow - up compared with subjects with higher levels of 25(oh)d ( > 15 ng / ml ) . in the health professionals follow - up study , men without prior cardiovascular disease and vitamin d levels of < 15 ng / ml showed a twofold increase in the rate of myocardial infarction . a cross - sectional study by dobnig et al . found an inverse relationship between lower levels of vitamin d and increased risk of all - cause and cardiovascular mortality . other cross - sectional observational studies have confirmed that lower vitamin d levels are associated with endothelial dysfunction as well as arterial stiffness . the vasoprotective action of vitamin d may be mediated by increasing nitric oxide ( no ) production , inhibiting foam cell formation , and reducing the expression of adhesion molecules in endothelial cells [ 1921 ] . the recognition of specific vdr genetic susceptibility in the pathophysiology of hypertension has further supported these insights . with regards to cerebrovascular disease , in a large population - based prospective study in copenhagen , an increase risk of symptomatic ischemic stroke was observed with decreasing plasma 25(oh)d concentrations . a similar association with vitamin d deficiency was also demonstrated in the honolulu heart program , which studied 7385 men over a 34-year period . in a study of 250 stroke patients in india , a deficiency of 25(oh)d studies are needed to establish if vitamin d supplementation reduces the risk of ischemic stroke in the general population . li et al . established that vdr knockout mice have elevated blood pressure , cardiac hypertrophy , and elevated activation of the renin angiotensin aldosterone system ( raas ) , which can be reversed with an angiotensin - converting enzyme ( ace ) inhibitor [ 26 , 27 ] . raas plays a pivotal role in maintaining sodium and blood volume homeostasis by modulating renal function and blood pressure . raas induces recruitment and activation of inflammatory cells within the vessel wall , promoting endothelial dysfunction and increasing vascular permeability . this inflammatory response stimulates hyperplasia and hypertrophy of vascular smooth muscle cells and the release of pro - inflammatory molecules ( vcam-1 , monocyte chemoattractant protein-1 , interleukins 6 and 8) . also showed that wild - type mice given an injection of 1,25(oh)2d3 demonstrated suppression of renin mrna expression [ 26 , 27 ] . vitamin d is a potent negative regulator of the raas system , which may play an important role in the development of neuropathy in diabetes . raas inhibition is one of the few proven therapies , which may prevent or delay diabetic peripheral neuropathy ( dpn ) , evidenced by experimental studies but also two human clinical trials [ 2931 ] . in an experimental model of type 1 diabetes mellitus , plasma renin activity levels were decreased whereas ace activity was increased in diabetic rats compared with controls . a small open - label study of lisinopril in hypertensive subjects ( n = 13 ) showed improvements in nerve conduction measures over 12 weeks and a double - blind randomized controlled study found that administration of the ace inhibitor trandolapril in normotensive patients with mild diabetic neuropathy resulted in an improvement in neurophysiology over 12 months . in a randomized controlled trial ( rct ) by trivedi et al . , vitamin d3 100,000 iu was supplemented every 4 months for 5 years versus placebo to assess fracture risk . there was a non - significant trend [ risk ratio ( rr ) , 0.84 ; 95% ci , 0.0551.10 ] toward a reduction in cardiovascular deaths . a further rct ( n = 302 ) added vitamin d to ongoing calcium supplementation with a primary endpoint of the risk of falls . they reported as adverse events ischemic heart disease event rates of 1.3% in those on vitamin d versus 2.0% for placebo ( only two versus three events ) . these two rcts were analyzed in a meta - analysis which reported no significant benefit for vitamin d supplementation . in the women s health initiative ( whi ) study ( clincialtrials.gov identifier , nct00000611 ) , 36,282 women were randomized to receive a combination of vitamin d3 400 iu and calcium 1000 mg per day or placebo . myocardial infarction or coronary heart disease death was confirmed for 499 women assigned to calcium / vitamin d and 475 women assigned to placebo ( hazard ratio , 1.04 ; 95% ci , 0.921.18 ) . it was concluded that calcium plus vitamin d supplementation neither increased nor decreased coronary or cerebrovascular risk in generally healthy postmenopausal women over a 7-year use period . vdr have also been identified in pancreatic beta cells , and vitamin d deficiency has been shown to impair insulin synthesis and secretion in animal models of diabetes , suggesting a role in the development of type 2 diabetes . this in turn has been linked to increased insulin resistance , which is associated with diabetes , hypertension , inflammation , and increased cardiovascular risk . several studies , including one published by scragg et al . , demonstrated that lower vitamin d levels were associated with an increased risk of diabetes . a double - blinded placebo trial by mitri et al . showed that in adults at risk of type 2 diabetes , short - term supplementation with cholecalciferol improved beta cell function and had a marginal effect on attenuating the rise in hba1c . furthermore , two studies have shown an inverse relationship of 25(oh)d with hba1c in subjects with and without diabetes [ 41 , 42 ] . . found that the first quartile of serum 25(oh)d level , compared with the fourth quartile , was associated with an increased adjusted odds ratio ( or ) for prediabetes and vitamin d levels were inversely correlated with fasting glucose ( r = 0.29 , p = 0.04 ) and homeostasis model assessment ( homa ) ( r = 0.34 , p = 0.04 ) . however , in the whi study , of 33,951 participants receiving calcium plus vitamin d3 supplementation , there was no reduction in the risk of developing diabetes over 7 years . vitamin d also has an immunomodulatory effect as suggested by a study of more than 10,000 finnish children who were given 2000 iu vitamin d3 per day during the first year of life and demonstrated an astounding 78% reduced risk of type 1 diabetes over a 30-year follow - up . a small placebo controlled trial of vitamin d3 ( 4000 iu / day for 6 months ) supplementation showed reduced insulin resistance in a south asian population who were vitamin d deficient . recently , in an observational study of patients in the 5-year fenofibrate intervention and event lowering in diabetes ( field ) trial , low blood 25(oh)d concentrations were associated with an increased risk of macrovascular and microvascular disease events in type 2 diabetes . a 25(oh)d concentration < 20 ng / ml had a higher cumulative incidence of macrovascular and microvascular events than those with levels 20 ng / ml . indeed severe vitamin d deficiency has been shown to predict not only mortality but the development of nephropathy and retinopathy in type 1 diabetes . previously studies of vitamin d have largely focused on bone health and calcium metabolism ; however , an increasing body of evidence particularly over the past two decades implicates a role of vitamin d in the nervous system [ 48 , 49 ] . multiple neurodegenerative diseases including multiple sclerosis [ 5054 ] and parkinson s disease , as well as cognitive decline in the elderly , have been linked with declining vitamin d status . there are some data to suggest that treatment with high doses of vitamin d3 slows down the progression of disability linked to multiple sclerosis ; however , further larger interventional studies are required to truly assess any therapeutic benefit . nerve growth factor ( ngf ) was first identified as being essential for the development of nociceptive primary neurons and later found to have a role in inflammatory hyperalgesia in adults . ngf is a neurotrophic factor which is synthesized initially as pro - ngf , prior to being cleaved by intracellular proteases before being translocated to the rough endoplasmic reticulum and is cleaved a second time by extracellular proteases to produce the active form . ngf is responsible for the development and maintenance of neurons in several regions in the central nervous system [ 59 , 60 ] . in addition , following neuronal injury , ngf has the ability to promote myelination of schwann cells , stimulate axonal sprouting , and guide axonal growth . brain barrier in experimental models and may act with relative specificity to increase ngf in glial cells and fibroblasts . vitamin d has been linked to the regulation of neurotrophins such as ngf and neuronal ca homeostasis , both of which may play a neuroprotective role in the peripheral nerve . showed that sciatic nerve ngf was preserved in animals exposed to a vitamin d analogue ( cb1093 ) whilst another vitamin d analogue ( mc903 ) has been shown to increase ngf synthesis . ngf is known to be depleted in experimental diabetes and in a study of patients with diabetic neuropathy a significant correlation was demonstrated between skin keratinocyte ngf immunostaining and skin axon - reflex vasodilation , mediated by small sensory fibers . stimulation of neurotrophin production by 1,25(oh)2d3 is correlated with a neuroprotective effect [ 66 , 70 ] and other than ngf , glial cell line - derived neurotrophic factor ( gdnf ) is also upregulated by 1,25(oh)2d3 . it is notable that in central nervous system tumors , 1,25(oh)2d3 and several synthetic analogues are effective in inducing a cell death pathway in glioma cells [ 7274 ] . importantly , topical application of ngf in diabetic foot ulcers has resulted in promotion of healing . ngf has previously been successfully studied in phase 2 clinical trials but showed a lack of efficacy in a phase 3 study for dpn . the prevalence of dpn can be as high as 50% and symptomatic diabetic neuropathy can affect approximately 30% of diabetic patients with neuropathy . currently there are no licensed therapies which may alter the natural history of dpn ; therefore , symptom control remains the mainstay of dpn management . even for symptom relief , effective treatment continues to remain a major challenge and anticonvulsants and antidepressants are first - line therapy with a 50% reduction in pain considered a good outcome . in the largest observational study to date of diabetic patients in a primary care cohort in the uk ( n = 15,692 ) , the prevalence of painful symptoms and additionally the adjusted risk of painful neuropathic symptoms in type 2 diabetes was double that of type 1 diabetes ( or , 2.1 ; 95% ci , 1.72.4 ; p < 0.001 ) and women had a 50% increased adjusted risk of painful symptoms compared with men ( or , 1.5 ; 95% ci , 1.41.6 ; p < 0.0001 ) . furthermore , despite a lower prevalence of neuropathy in south asians ( 14% ) compared to europeans ( 22% ) and african caribbeans ( 21% ; p < 0.0001 ) , painful symptoms were greatest in south asians ( 38% vs. 34% vs. 32% , p < 0.0001 ) . south asians without neuropathy maintained a 50% increased risk of painful neuropathic symptoms compared with other ethnic groups ( p < 0.0001 ) . these differences may partly be explained in relation to vitamin d deficiency as these groups have been shown to have excess vitamin d deficiency . other studies have assessed ethnic differences in dpn in a us population with variations noted in risk for dpn and diabetic autonomic neuropathy . however , in an australian study there were no significant differences between ethnic groups although the ethnic mix of the last two studies differs from that in the uk . an increasing body of data suggests that vitamin d may have not only analgesic properties but also additional neuroprotective benefits . vitamin d deficiency and insufficiency are associated with various pain syndromes and low levels of vitamin d correlated with the presence of peripheral neuropathy in primary sjgren s syndrome which can lead to a small fiber neuropathy . vitamin d supplementation has been shown to improve musculoskeletal pain and non - traumatic back pain in some studies . however , there is a prominent contrast in treatment effects between randomized , double - blind trials that minimized bias and those with designs known to be subject to bias with better treatment effects noted in the latter . therefore , the overall evidence for the use of vitamin d for chronic pain in adults is poor because of low - quality and insufficient rcts . pain thresholds of multiple etiologies have been reported to be lowered with vitamin d deficiency and elevated when the vitamin d deficiency is corrected . previous observational studies have demonstrated a significant link between vitamin d deficiency and dpn [ 8991 ] . in the national health and nutrition examination survey ( nhanes ) 20012004 , an unweighted sample of 591 subjects with diabetes demonstrated a significant association between vitamin d deficiency and both paresthesia ( or , 2.12 ; 95% ci , 1.173.85 ) and numbness ( or , 2.04 ; 95% ci , 1.183.52 ) after adjusting for obesity , co - morbidities , use of medications for neuropathy , diabetes duration , and glycemic furthermore , in a prospective observational study conducted by skalli et al . in 111 consecutive ambulatory patients with type 2 diabetes an association between vitamin d deficiency and peripheral diabetic neuropathy was found . although both subjects with and without dpn were deficient , those with dpn had approximately 4 ng / ml lower 25(oh)d levels . in a further cross - sectional study by shehab et al . , vitamin d deficiency was an independent risk factor for dpn assessed using the neuropathy symptom score and other clinical and electrophysiological measures of dpn . the duration of diabetes was higher in the group with peripheral neuropathy as was elevated low - density lipoprotein - cholesterol ( ldl ) . this latter observation may be of relevance as both vitamin d and cholesterol share a common metabolic pathway through 7-dehydrocholestrol . in a more recent study conducted in a small homogenous population in rural turkey , reduced serum 25(oh)d was associated with dpn ; interestingly , neither vdr nor vdr - binding protein differed between those with and without dpn . large nerve fiber deficits have also been associated with vitamin d status in diabetes mellitus . in an age , sex , body mass index ( bmi ) , height , and disease duration matched cohort ( n = 33 ) of patients with diabetes , 25(oh)d was significantly lower in the dpn group ( 21.2 11.5 vs. 13.5 5.1 ng / ml , p = 0.001 ) . after further adjustment for all studied variables , serum vitamin d had an independent and inverse association with both dpn presence and severity , as each 1 ng / ml increase in serum 25(oh)d was correlated with 2.2% and 3.4% decrease in the presence and severity of nerve conduction studies ( ncv ) impairment , respectively . in a meta - analysis of six studies that involved a total of 1484 patients with type 2 diabetes , vitamin d deficiency ( < 20 ng / ml ) was significantly associated with an increased risk of dpn . the or was 2.88 ( 95% ci , 1.844.50 ; p < 0.00001 ) with an adjusted or of 2.68 ( 95% ci , 1.674.30 ; p < 0.0001 ) in a subanalysis of three studies , suggesting that vitamin d deficiency is very likely to be associated with dpn in type 2 diabetic patients . of course , hypertension itself is an independent risk factor for dpn [ 95 , 96 ] and the interaction between vitamin d and the raas may provide a further link . to date clinical trials of vitamin d intervention for dpn are limited . a single case report has shown reversal of symptoms in severe intractable painful diabetic neuropathy with the administration of 50,000 iu ergocalciferol ( vitamin d2 ) weekly with almost complete resolution by 4 weeks . despite the fact that the patient was not severely deficient with a baseline 25(oh)d of approximately 16 ng / ml , the subsequent pain relief from the weekly ergocalciferol dose enabled the discontinuation of oxycodone . conducted an open - label uncontrolled study in patients with vitamin d deficiency and 51 patients with painful dpn . after treatment with oral cholecalciferol ( vitamin d3 ) at a mean dose of 2059 iu daily for 3 months there was an improvement of approximately 50% on the visual analogue scale ( pain ) . although the placebo effect can be considerable in trials of therapeutic agents in painful dpn , the maximal placebo effect expected would be no more than approximately 30% . indeed a 50% pain reduction is considered to be efficacious when evaluating interventions in painful dpn . conducted a double - blind rct of a compound ( qr-333 ) containing a vitamin d analogue and showed positive effects on numbness , jolting pain , and irritation in subjects with painful dpn . however , the outcomes of this study are somewhat confounded as qr-333 contained quercetin , a flavonoid with aldose reductase inhibitor effects , which is one of the key pathogenetic pathways causing diabetic neuropathy . hard endpoints of dpn is foot ulceration , which may eventually lead to lower limb amputation and significant morbidity and mortality . in a cross - sectional study of diabetic patients without ( n = 162 ) and with ( n = 162 ) plantar ulcers , lower 25(oh)d status was associated with plantar ulceration in patients with diabetes ( 6.3 vs. 28 ng / ml , p < 0.005 ) . in a further study by tiwari et al . , vitamin d deficiency was more prevalent and severe in patients with diabetic foot infection . cases of foot infection ( n = 125 ) had a greater risk of vitamin d deficiency ( 25(oh)d < 20 ng / ml ) than controls ( n = 164 ) with an or of 1.8 ( 95% ci , 1.13.0 ; p = 0.02 ) and the risk of severe vitamin d deficiency ( 25(oh)d < 10 ng / ml ) was significantly higher in cases than in controls with an or of 4.0 ( 95% ci , 2.46.9 ; p < 0.0001 ) . patients with chronic kidney disease ( ckd ) are known to have deficiency of both 25(oh)d and 1,25(oh)2d , which is associated with a high cardiovascular mortality . although in patients with ckd , higher levels of vitamin d may also be associated with vascular calcification . experimental studies have shown a role of the vdr , and vdr - mediated vitamin d actions are thought to be renoprotective in diabetic nephropathy . in an experimental study , vitamin d / vdr signaling in podocytes played a critical role in the protection of the kidney from diabetic injury . analysis of the nhahes ( 20012006 ) cross - sectional data showed that there was an independent association between vitamin d deficiency and insufficiency with the presence of nephropathy . in a small double - blind , randomized placebo - controlled , crossover trial of paracalcitriol ( a vitamin d analogue ) in patients with proteinuria despite adequate raas blockade , the urinary albumin excretion rate was significantly further lowered compared to placebo . furthermore , combination therapy with an at1 blocker and a vitamin d analogue markedly ameliorated diabetic nephropathy in an experimental model of diabetic nephropathy . in the vital study ( clinicaltrials.gov identifier , nct00421733 ) , a placebo - controlled , double - blind rct , paracalcitriol ( a vitamin d analogue ) added to raas inhibition at a dose of 2 g / day significantly lowered blood pressure and residual albuminuria in patients with diabetic nephropathy , further highlighting the role of vitamin d as a potent raas inhibitor [ 113 , 114 ] . recently , a systematic review and meta - analysis by derakhshanian et al . suggested that despite a higher risk for nephropathy in vitamin d - deficient patients with diabetes , vitamin d supplementation did not support causality in this association . a pooled data of urinary albumin creatinine ratio levels in clinical trials ( five trials , however , these studies were small and heterogeneous and the vitamin d replacement dose was small . thus , larger well - designed rcts with adequate vitamin d supplementation in diabetic nephropathy are required . 25(oh)d concentrations are associated with optic chiasm volume and several studies have found an association between vitamin d deficiency and age - related macular degeneration ( amd ) [ 117 , 118 ] . in a mouse model of ischemic retinopathy , 1,25(oh)2d was shown to inhibit neovascularization in retinal tissue . in a turkish cohort of 66 subjects , aksoy et al . demonstrated an inverse correlation between worsening diabetic retinopathy and lower 1,25-dihydroxyvitamin d3 ( active vitamin d ) . in 50 diabetic patients with early - stage diabetic retinopathy ( dr ) with vitamin d deficiency and 50 early - stage dr without vitamin d deficiency , vitamin d deficiency was associated with early retinal nerve fibre layer ( rnfl ) thinning . furthermore , in a korean study sampling 18,363 from the nhanes ( 20082012 ) , 25(oh)d was associated with diabetic retinopathy . similar results have been replicated in a chinese population with type 2 diabetes and a japanese population with type 1 diabetes . however , in the third nhanes , no relationship was found between 25(oh)d levels and retinopathy severity . also the eurodiab prospective complications study showed no significant relationship between 25(oh)d and retinopathy , despite a positive relationship with microalbuminuria . indeed , most recently , alam et al . have shown no association between serum 25(oh)d concentrations and the presence and severity of diabetic retinopathy or maculopathy ; however , the marked deficiency in their population may have confounded the study as approximately 91% of subjects were deficient / insufficient ( 25(oh)d < 30 ng / ml ) . the prevalence of vitamin d deficiency is in pandemic proportions in the uk and worldwide , particularly in the diabetic population . an increasing body of literature suggests a possible pathogenetic role of vitamin d in the long - term complications of diabetes and vitamin d deficiency may also exacerbate symptoms of painful dpn . despite a number of well - designed observational studies a causal link remains to be demonstrated . the association between vitamin d status and cardiometabolic outcomes is uncertain , especially as intervention trials have shown no clinically significant effect of vitamin d supplementation . however , short - term underpowered interventions with low doses of vitamin d may have produced limited benefits . given the widespread propensity to deficiency and its low side effect profile , vitamin d therapy represents a promising therapeutic intervention in the treatment of diabetic complications . in particular , there is a significant need for good quality rcts using therapeutic doses ( approximately 4000 iu / day ) of vitamin d in painful dpn .
we describe a 64-year - old male patient with panhypopituitarism who experienced polymorphic ventricular tachycardia ( vt ) associated with long qt intervals . the panhypopituitarism developed as a sequelae of radiation therapy administered 20 years prior to his current presentation and was recently aggravated by urinary tract infection with sepsis . in this case , polymorphic vt was resistant to conventional therapy ( including magnesium infusion ) , and qt prolongation and t wave inversion were normalized after the administration of steroid and thyroid hormones . thyroid hormone is generally known to be associated with torsades de pointes ( tdp ) , but steroid or other hormones may also provoke tdp . hormonal disorders should be considered as a cause of polymorphic vt with long qt intervals . some arrhythmias can be life - threatening , and they can be prevented with supplementation of the insufficient hormone .	torsades de pointes ( tdp ) may occur as a consequence of acquired qt interval prolongation , resulting from the use of various medications , liquid protein diets , intracranial events , bradyarrhythmias , electrolyte disturbances , and hormonal disorders.1 - 4 ) because many cases of polymorphic ventricular tachycardia ( vt ) have been reported , and the condition is resistant to antiarrhythmic drug therapy , it is important to determine the primary cause of long qt intervals and the way to correct it . we experienced a case of tdp associated with panhypopituitarism , which developed as a sequelae of radiation therapy administered 20 years prior to the current presentation and was recently aggravated by urinary tract infection ( uti ) sepsis . a 64-year - old man was admitted to our hospital from shock caused by a uti . the patient had a medical history of local radiation therapy on the surgical area after undergoing functional endoscopic sinus surgery for a nasal tumor ( left maxillary cancer , squamous cell carcinoma ) 20 years ago . he was followed up for 5 years after the surgery , but the follow - up was discontinued , as there was no possibility of recurrence . he took tamsulosin for benign prostatic hypertrophy and used spiriva ( spiriva , boehringer ingelheim gmbh , germany ) inhaler for chronic obstructive pulmonary disease . at admission , the patient 's blood pressure was 70/40 mm hg , his pulse rate was 88 beats / min , his respiration rate was 26 times per minute , and his body temperature was 38.0. the patient was managed for septic shock with antibiotics , including ceftazidime , in the intensive care unit ( icu ) , and his blood pressure stabilized . on the 4th day of icu admission , non - sustained polymorphic vt ( tdp ) occurred for 20 seconds . the patient indicated that he briefly felt drowsy , but his blood pressure after the event was 110/70 mm hg . laboratory tests reported the following findings : glucose 141 mg / dl ( 70 - 110 mg / dl ) ; na 144 meq / l ( 135 - 145 meq / l ) ; k 3.8 meq / l ( 3.5 - 5.5 meq / l ) ; corrected ca 8.6 mg / dl ( 8.4 - 10.2 mg / dl ) ; and mg 1.4 mg / dl ( 1.9 - 2.5 mg / dl ) . the patient was treated with a loading dose of mg ( 2 g ) and a maintenance dose of mg ( 1 g / day for 8 days ) . after the administration of magnesium , the patient 's laboratory parameters were as follows : na 144 meq / l ( 135 - 145 meq / l ) ; k 4.2 meq / l ( 3.5 - 5.5 meq / l ) ; corrected ca 8.8 mg / dl ( 8.4 - 10.2 mg / dl ) ; and mg 2.4 mg / dl ( 1.9 - 2.5 mg / dl ) . although his mg level normalized , the qtc interval prolongation and t wave inversion persisted , and tdp recurred several times . when tdp appeared , the patient 's blood pressure was stable , but he became stuporous . therefore , echocardiography , coronary angiography , and temporary ventricular overriding pacing with a ventricular rate of 90 beats per minute were performed . echocardiography demonstrated a left ventricular ejection fraction of 54% with no regional wall motion abnormality , and no abnormal findings were observed during coronary angiography . a thyroid function test showed reduced values : t3 35 ng / dl ( 65 - 150 ng / dl ) ; free t4 0.36 ng / dl ( 0.78 - 1.54 ng / dl ) ; and thyroid - stimulating hormone ( tsh ) 0.27 iu / ml ( 0.35 - 5.5 iu / ml ) . luteinizing hormone ( lh ) and follicle stimulating hormone ( fsh ) were measured to evaluate abnormal thyroid functioning . the tsh releasing hormone stress test demonstrated no increase in tsh ( 0.22 iu / ml ; range 0.35 - 5.5 iu / ml ) , lh ( 0.9 miu / ml ; range 1.0 - 5.3 miu / ml ) ; and fsh ( 72.5 miu / ml ; range 1.1 - 13.5 miu / ml ) . after panhypopituitarism was diagnosed , brain ct and brain mri with enhancement were performed , and a small pituitary gland was found . before treatment , the average urine output was 4 l daily . the patient 's serum and urine osmolarity were 300 mosm / kg ( 275 - 295 mosm / kg ) and 247 mosm / kg ( 300 - 900 mosm / kg ) , respectively . to correct the pituitary gland dysfunction , a steroid ( prednisolone , 7.5 mg ) , thyroid hormone ( levothyroxine , 100 g ) , and desmopressin ( minirin nasal spray , 5 g bid ) were administered . after one week of hormone treatment , the qtc prolongation and t wave inversion decreased mildly . after 4 weeks of steroid hormone , thyroid hormone , and desmopressin treatment , electrocardiography showed a normal qtc range ( 430 ms ) , and the t wave inversion disappeared ( table 1 ) . after desmopressin administration , the patient 's symptom of diabetes insipidus improved , and his urine output decreased to 1 - 2 l daily . his serum osmolarity normalized to 288 mosm / kg ( 275 - 295 mosm / kg ) , and his urine osmolarity increased to 544 mosm / kg ( 300 - 900 mosm / kg ) . the physiological chronotropic response and normal tension of the heart muscle in the diastolic phase depend on the proper expression of tri - iodothyronine in the heart cells and its stimulating influence on na - k atpase and ca atpase in the endoplasmic reticulum . normal heart contractility is also related to proper tri - iodothyronine - stimulated transcription of the myosin heavy - chain alpha gene and inhibition of the heavy - chain beta gene . moreover , proper tri - iodothyronine expression in the cardiac muscle affects the number of -adrenergic receptors and their sensitivity to catecholamines . profound hypothyroidism and decreased expression of tri - iodothyronine in the heart cells may cause a worsening of cardiac contractility , a decreasing heart rate and a slowing of the conduction of electrical stimuli in the heart muscle . this may be the reason for bradycardia and prolongation of the qt interval . as a result , life threatening arrhythmias may occur , such as torsade des pointes - type tachycardia.5)6 ) in addition to thyroid hormone , steroid hormones are also known to be associated with tdp.7 - 9 ) although the pathogenesis remains unknown , hypopituitarism has been reported to be commonly associated with electrocardiac abnormalities . t wave inversion was the most common finding , and prolongation of the qt interval was the second most common finding.10 ) with ecg changes , there were some reports of polymorphic vt with hypopituitarism . although the mechanism remains unclear , with glucocorticoid deficiency , an intracellular and extracellular electrolyte imbalance in myocytes and histopathological changes in the myocardium are thought to play a role in this disorder . it was reported that glucocorticoids up - regulate kv 1.5 k channel gene expression in the rat ventricle.11 ) additionally , iga et al.12 ) suggested that catecholamine release induced by hypoglycemia might cause arrhythmia or abnormal wall motion of the left ventricle in patients with adrenal insufficiency . because it is known that the female gender is associated with a higher risk for polymorphic vt and sudden cardiac death in adult patients with congenital long qt syndrome ( lqts)-type 1 and type 2 , there is a potential role of the sex hormones in modulating the arrhythmogenic risk in lqts.13 ) a previous animal study indicated that the occurrence of vt and sudden cardiac death was abolished with oral progestins in transgenic lqt2 rabbit model.14 ) however , there was no clear relationship between tdp and sex hormone and acquired lqts . in our case , we were unable to determine which hormonal effect was the most important , because thyroid hormone and glucocorticoid hormone were administered together . to the best of our knowledge , this is the only report of an association between tdp and panhypopituitarism that developed as a sequelae of radiation therapy . previous reports described cases of tdp with hypopituitarism , caused by sheehan syndrome and the sequelae of hemorrhagic fever with renal syndrome.8)9 ) with the administration of steroid hormone and thyroid hormone , the patient 's qtc prolongation and inverted t wave showed improvement , and tdp did not recur . hormonal disorders should be considered as a cause of polymorphic vt with long qt intervals . with supplementation of the deficient hormones , this life - threatening arrhythmia can be cured .
realizing the vast technological potential of patternable block copolymers requires both the precise controlling of the orientation and long - range ordering , which is still a challenging topic so far . recently , we have demonstrated that ordered nanoporous thin film can be fabricated from a simple supramolecular assembly approach . here we will extend this approach and provide a general route to fabricate large areas of highly ordered polymeric nanodot and nanowire arrays . we revealed that under a mixture solvent annealing atmosphere , a near - defect - free nanoporous thin film over large areas can be achieved . under the direction of interpolymer hydrogen bonding and capillary action of nanopores , this ordered porous nanotemplate can be properly filled with phenolic resin precursor , followed by curation and pyrolysis at middle temperature to remove the nanotemplate , a perfect ordered polymer nanodot arrays replication was obtained . the orientation of the supramolecular assembly thin films can be readily re - aligned parallel to the substrate upon exposure to chloroform vapor , so this facile nanotemplate replica method can be further extend to generate large areas of polymeric nanowire arrays . thus , we achieved a successful sub-30 nm patterns nanotemplates transfer methodology for fabricating polymeric nanopattern arrays with highly ordered structure and tunable morphologies .	precise fabrication of large areas of ordered nanoscale structures is essential for microelectronic and information technology , the broad scope of top - down processes , including conventional immersion lithography , extreme ultraviolet lithography , and soft lithography have been proposed to meet the demands of devices miniaturization , these endeavors have enabled the lateral dimensions of devices to be readily shrunk below 100 nm [ 1 - 3 ] . however , as the lateral dimension goes smaller and smaller , these top - down approaches become extremely difficult and expensive , hence , other methodologies of creating nanostructures are of great interesting if they can offer advantages in reduced production cost , smaller feature sizes , and more complex nanopatterns . nanofabrication via block copolymer self assembly represents one of the most powerful candidates , and is now taking as the most promising methodology for next - generation lithography [ 4 - 6 ] , mainly due to their intrinsic nanoscale dimensions , facile synthesis , and strict control of architecture . ever since the self assembly of block copolymers was introduced as a powerful bottom - up route to well - organized nanostructures decade ago , many efforts have been devoted : as through chemical modification of the block copolymer structure to achieve special functionalities , exploring electric fields , and interfacial interactions to control the orientation , and utilizing solvent induced ordering , salt complexes , and shear fields to achieve ordered arrays [ 7 - 10 ] . among which solvent annealing is of particularly beneficial mainly due to their mild process condition and no need for additional complicated apparatus , and now it has turn out to be a very simple while robust approach to generate almost defect - free microphase separation structures in bcp thin films [ 11 - 13 ] . even more , it appears to be the single possible way for thermal lible systems such supramolecular block copolymers based on noncovalently bonding . further research revealed the use of a co - solvent atmosphere , will enables one to enhancing the ordering process ever further . however , a critical drawback of solvent annealing is that bcp thin film often de - wets its substrate during solvent exposure as have been already pointed out by several researchers [ 16 - 19 ] . this makes it is very difficult to obtain uniform and ordered bcp thin film over a macroscopic area without the direction of additional external fields . in many cases , realizing the vast technological potential of block copolymers requires both the precise controlling of the orientation and long - range ordering , however , weakness still remains , so far , only few works have reported to achieve highly ordered thin film nanotemplates , and the control of well - organized structures over large scale is still a challenging topic . in recent decade , ikkala and ten brinke have thoroughly demonstrated that well - ordered nanostructures in the bulk may be fabricated through supramolecular assemblies ( sma ) of low molecular amphiphiles and block copolymers . the amphiphiles can be physically bonded to homopolymers and block copolymers using noncovalent interactions , this complexation can lead to the formation of supramolcecular block copolymer which can further assembled into hierarchy nanostructures with various responsive properties . more recently , we have demonstrated that ordered nanoporous thin film can be fabricated from a similar approach based on the supramolecular assemblies of block copolymers ps - pvp and small molecule ( 2,4-hydroxybenzeneazo benzoic acid , haba ) [ 22 - 26 ] , the sma thin films demonstrated hexagonal cylindrical morphology with ps form the matrix . solvent annealing in dioxane can enhance the ordering of thin films microphase separation , following extraction of haba with selective solvent methanol results in a nanoporous thin films . the channels can be filled with metal , for example , nickel , by electrochemical deposition to fabricate an array of ordered metal nanodots or nanowires with some defects appear in the array due to the nonuniform electrodeposition kinetics of the metal clusters in nanochannels . in this article , we further investigate the ps - pvp / haba supramolecular assembly system in order to achieve a highly ordered morphology and to explore a high definition nanotemplate replication method for fabrication of highly ordered polymeric nanodots and nanowire arrays . we will demonstrate that under a mixture solvent annealing atmosphere ( with the dedicating choosing of an additional nitromethane as a selective solvent for the minor component ) , a near - defect - free nanoporous thin films with long - range ordering over a large areas can be achieved . taking aim at high definition nanotemplate transfer technique which is another daunting obstacle to the application of the nanoporous template , we will further show that under the direction of the capillary action and hydrogen bonding , this ordered nanoporous template can be perfectly transferred , and thus achieved a methodology for the preparation of highly ordered sub-30 nm polymeric nanodot and nanowire arrays . poly(styrene - block-4-vinylpyridine ) ( ps - pvp ) , with mn ps 4000 g / mol , pvp 5600 g / mol , mw / mn 1.06 ) for both blocks , was purchased from polymer source inc . a soluble low - molecular - weight phenolic resin precursor solution was prepared from phenol and formaldehyde using a basic polymerization method . solvents 1,4-dioxane , chloroform , methanol , and dichloromethanes were purchased from acros organics and used as supplied . ps - pvp and haba ( 1 mol of haba and 1 mol of 4-vinylpyridine monomer unit ) were dissolved separately in 1,4-dioxane . ps - pvp solution was slowly added dropwise to haba solution while heating to 95 c in an ultrasonic bath . additional 1,4-dioxane / nitromethane mixture solvent vapor annealing of a thin film was applied to improve the order of nanodomains . alternatively , the samples were treated in vapors of chloroform to arrange parallel alignment of the nanodomains . the nanoporous template was dip coating from the ethanol solution of phenolic resin precursor , the thin film was sequentially cured by exposure to formaldehyde gas at 100 c for 4 h. the cured film was finally pyrolysis at middle temperature ( heating to 450 c in 2 h and keep 2 h ) , to remove the ps - pvp and resulted ordered nanodots arrays ( fig . scheme of the fabrication of highly ordered polymeric nanodot and nanowire arrays templated by nanoporous thin films the thickness of the polymer films was measured by a se400 ellipsometer ( sentech instruments gmbh , germany ) with a 632.8 nm laser at a 70 incident angle . atomic force microscopy ( afm ) imaging was performed using a dimension 3100 scanning force microscope ( digital instruments , inc . analysis of the afm images ( fast fourier transform ) was performed with wsxm software ( nanotec electronica ) . poly(styrene - block-4-vinylpyridine ) ( ps - pvp ) , with mn ps 4000 g / mol , pvp 5600 g / mol , mw / mn 1.06 ) for both blocks , was purchased from polymer source inc . a soluble low - molecular - weight phenolic resin precursor solution was prepared from phenol and formaldehyde using a basic polymerization method . solvents 1,4-dioxane , chloroform , methanol , and dichloromethanes were purchased from acros organics and used as supplied . ps - pvp and haba ( 1 mol of haba and 1 mol of 4-vinylpyridine monomer unit ) were dissolved separately in 1,4-dioxane . ps - pvp solution was slowly added dropwise to haba solution while heating to 95 c in an ultrasonic bath . additional 1,4-dioxane / nitromethane mixture solvent vapor annealing of a thin film was applied to improve the order of nanodomains . alternatively , the samples were treated in vapors of chloroform to arrange parallel alignment of the nanodomains . the nanoporous template was dip coating from the ethanol solution of phenolic resin precursor , the thin film was sequentially cured by exposure to formaldehyde gas at 100 c for 4 h. the cured film was finally pyrolysis at middle temperature ( heating to 450 c in 2 h and keep 2 h ) , to remove the ps - pvp and resulted ordered nanodots arrays ( fig . 1 ) . scheme of the fabrication of highly ordered polymeric nanodot and nanowire arrays templated by nanoporous thin films the thickness of the polymer films was measured by a se400 ellipsometer ( sentech instruments gmbh , germany ) with a 632.8 nm laser at a 70 incident angle . atomic force microscopy ( afm ) imaging was performed using a dimension 3100 scanning force microscope ( digital instruments , inc . analysis of the afm images ( fast fourier transform ) was performed with wsxm software ( nanotec electronica ) . the as - deposited ps - pvp / haba thin film form cylindrical phase separation normal to the substrate , with poor lateral ordering . due to the thermal liability of hydrogen bonding , solvent vapor annealing is an elegant approach to promote bcp ordering , in the previous study , dioxane was chosen as the annealing solvent , here we dedicating chosen a 1,4-dioxane / nitromethane mixture solvent annealing in order to further enhances the long - range ordering in relatively short time . after being annealed in a dioxane / nitromethane mixture solvent for about 24 h , the films were taken out and rinsed in methanol for 5 min to destroy the hydrogen bonding and removes selectively haba from thin film , and resulted a nanoporous thin film , the afm revealed the orientation order is only of short - range , this is also apparent from a smashed ring in the fft plot ( fig . the inset is the fft plot of the image however , further prolonging solvent annealing time to about 72 h in mixture atmosphere lead to a dramatically increase in the long - range ordering of the thin film , afm image clearly identified a near - defect - free ordered arrays of highly ordered hexagonal structure with all pores oriented perpendicular to the substrate ( fig . 3 ) , the fourier transform plot of the corresponding afm phase image is shown in the inset , the six sharp first - order peaks clearly indicate the presence of a highly ordered hexagonal structures , this higher order peaks attest to the high degree of order within the thin film . the mean center - to - center distance of the nanopores based on the afm image is about 30 nm . during solvent annealing , ps , and p4vp / haba , blocks are swelled by dioxane vapor and tend to organize into a ordered structures , however , this process is restrained by dioxane in a certain extent due to the good solubility of dioxane for all the blocks , with the addition of nitromethane which is a good solvent only for the minor pvp / haba block , the repulsion between the ps and p4vp / haba domains is enhanced , a fast and highly ordered defect - free microphase separation structure is achieved . afm images of the nanoporous thin filmsaheight image , bphase image , cenlarged height image . mixture solvent annealing 72 h , and washed with methanol . the inset is the fft plot of the afm image these nanoporous thin films can be used as scaffolds for fabricating organic , inorganic , and metal nanostructures . compared with the well studied inorganic and metal nanostructures , organic ( polymeric ) nanostructures remains less researched , despite their great potential in catalysis and membrane separation . here , we demonstrated that highly ordered phenolic resin nanodot and nanowire arrays can be prepared through perfect replication of the ordered nanotemplate with the direction of hydrogen bonding and the capillary action of nanopores . the above prepared nanoporous thin films were immersed in the phenolic resin precursor / ethanol solution , the nanopores were immediately filled by phenolic precursors due to the capillary action of cylindrical nanopores the formation of interpolymer hydrogen bonding complex between the pvp in the inner pore surface of the nanotemplate and the phenolic resin . as ethanol is just a good solvent for minor component pvp , while is a nonsolvent for the matrix composed of ps , thus , the nanoporous templates are maintained during the dip coating process . when the template was drop from the precursor solution , the ethanol vaporized and the phenolic resin precursor were maintaining inside the nanopores . after curation with formaldehyde , the filled nanotemplates were then pyrolysed at middle temperature ( heating to 450 c in 2 h and keep 2 h ) , this temperature is enough to degrade ps - pvp nanotemplate , while the phenolic resin can still maintained . afm height image revealed , after pyrolysis , highly ordered discrete phenolic resin nanodots arrays with uniform diameter can be observed ( fig . 4 ) , of special interest is that the original highly ordered structure of nanoporous thin film was almost maintained throughout the nanotemplate transfer process and thus resulted a perfect nanotemplate transformation ; the average center - to - center distance between the nanodots was 30 nm , which was identical to that of the original porous nanotemplate . afm images of highly ordered polymeric nanodots arrays from nanoporous thin film after pyrolysis.aheight image , bphase image . lateral scale 1500 nm 1500 nm.cenlarged height image in addition to preparation of nanodots arrays , we further extend this facile nanotemplate replica method to fabricate polymeric nanowire arrays . a special advantage of the supramolecular assembled ps - pvp / haba system we used here is that the orientation of the phase separation can be reversibly switched from the perpendicular to parallel orientation and vice versa upon exposure to 1,4-dioxane or chloroform vapor , respectively . thus , instead of dioxane vapor , the ps - pvp / haba supramolecular assembly thin film was annealed in a saturated chloroform vapor for short time of 15 min to achieve re - alignment . after rinsing with methanol , the afm revealed that most of cylindrical microdomains orientation was re - alignment parallel to the substrate , still it was clear that 15 min is not enough to fulfill the re - alignment process and thus the coexistence of normal and parallel alignments of nanodomains were observed ( fig . 5a ) . followed the same template transfer process , this nanotemplate was dip coating from phenolic resin precursor solution , and followed by curation and middle temperature pyrolysis , large areas of short polymeric nanowires with nanodots mixture were formed ( fig . aafm height images of thin film nanotemplate , after re - alignment using chloroform annealing for 15 min.bheight images of the resulted short polymeric nanowire arrays replica after pyrolysis . lateral scale 2000 nm 2000 nm further prolonging the chloroform solvent annealing time to 30 min is enough to fulfill the re - alignment transformation process , after rinsing with methanol , the afm revealed that all the cylindrical microdomains were oriented parallel to the substrate(fig . 6a ) , and thus after deposition and middle temperature pyrolysis , a longer nanowire arrays which was identical to that in the original nanotemplate can be achieved ( fig . aafm height images of thin film nanotemplate , after re - alignment using chloroform annealing for 30 min.bafm height images of polymeric nanowire arrays replica after pyrolysis . in summary , we have provided a general route to fabricate highly ordered polymeric nanodot and nanowire arrays using supramolecular assembled block copolymer thin film as nanotemplates through the interpolymer hydrogen bonding capillary action . the as - deposited ps - pvp / haba thin film formed randomly hexagonally packed cylindrical phase separation structures , with the dedicating choosing of mixture solvent annealing atmosphere ( dioxane as good solvent for both blocks and an additional nitromethane as a selective solvent only for the minor component pvp / haba ) , a near - defect - free nanoporous thin film with long - range ordering over a broader range of length scales can be achieved , extraction of haba microdomains resulted in highly ordered nanoporous thin films . under the direction of interpolymer hydrogen bonding and the capillary action of nanopores , the nanotemplate can be properly filled with phenolic resin precursor , followed by curation and pyrolysis at middle temperature which will selectively degrade the ps - pvp block copolymer nanotemplate , a perfect ordered nanodot arrays replication was obtained , thus resulted in an excellent and efficient transformation of the nanoporous template to functional polymeric nanodot arrays . the orientation of the supramolecular assembly thin films can be readily re - alignment from the perpendicular to parallel the substrate upon exposure to chloroform vapor , and thus this facile nanotemplate replica method can be further extend to generate large areas of polymeric nanowire arrays . thus , we have achieved a successful sub-30 nm patterns nanotemplates transfer methodology for fabricating polymeric nanopattern arrays with tunable morphology and lateral spacings .
physical fitness is considered a major factor contributing to the maintenance of independent living and everyday competence . in line with this notion , it has been shown that several years of amateur dancing experience can exert beneficial effects not only on balance and posture but also on tactile , motor , and cognitive functions in older people . this raises the question of whether an even more extensive schedule of dancing , including competitive tournaments , would further enhance these positive effects . we therefore assessed posture , balance , and reaction times , as well as motor , tactile , and cognitive performance in older expert ballroom dancers with several years of competitive experience . we found substantially better performance in the expert group than in the controls in terms of expertise - related domains like posture , balance , and reaction times . however , there was no generalization of positive effects to those domains that were found to be improved in amateur dancers , such as tactile and cognitive performance , suggesting that there might be an optimal range of intervention intensity to maintain health and independence throughout the human lifespan .	in addition to a general decline in physical fitness , the aging process is accompanied by a progressive decline in perception , motor behavior , cognition , and memory functions [ 24 ] . therefore , the preservation of everyday life skills and the maintenance of independent living become increasingly important with advancing age . it is well established that physical fitness is intimately associated with cognitive performance in the elderly [ 59 ] . consequently , high levels of physical fitness have been assumed to be a major factor contributing to the maintenance of independent living and everyday competence . one of the basic accomplishments of gerontology is the recognition of the tremendous heterogeneity and interindividual variability in the elderly . thus , the emergence of age - related decline can be highly variable between individuals , and there are notable differences in the interindividual performance of general skills at advanced ages . it seems that , aged individuals can maintain high levels of proficiency in certain domains involving cognitive - motor functions such as golf or piano playing . this gave rise to an intriguing question : how is proficiency in one domain of expertise , like playing piano , associated with performance in general ? older experts show little or no age - related decline in tasks related to their area of expertise , but beyond that they show a general age - related decline similar to the nonexpert older adults . on the other hand , the maintenance of high levels of expertise in one domain can have a positive impact on related functions . for example , elderly professional pianists have higher finger - tapping rates than untrained aged - matched individuals . we recently showed that a regular schedule of amateur dancing over many years throughout old age not only promotes posture and balance , but also has a wide range of beneficial effects on reaction times ( rts ) , motor behavior , and tactile and cognitive performance by comparing such individuals with an aged - matched nondancer control group . we hypothesized that the generalization of superior performance associated with regular dancing develops as a result of physical exercise in combination with cognitive challenges , sensory stimulation , and social interaction , all of which contribute to neuroplasticity . here , we extended these studies by investigating the impact of dancing at a higher level of expertise . one of the rationales for this study was to obtain information about whether a more extensive schedule of dancing , including competitive tournaments , would further enhance the range or magnitude of beneficial effects . we compared a group of neurologically healthy older subjects with many years of expert and competitive experience of dancing ( ed ) to a gender- , age- , and education - matched nondancer control group ( cg ) . in this study , the term expert is defined as those who regularly attend dance competitions and dance contests and undergo training at intensities of more than 4 h / week . comparable to our previous study on amateur dancers , we measured posture and balance , cognitive , attentional , intellectual , perceptual , and sensorimotor performance . subjects were recruited by advertisements in newspapers , poster announcements , and word - of - mouth advertising . all subjects reported their medical history and current medication and underwent the mini - mental status examination ( mmse ) . the ed group ( n = 11 , 5 women , 71.18 1.13 years ) had an extended history of competitive dancing ( 22.09 3.39 years ) with an average workload of 4.55 0.15 h / week . subjects in the ed group reported a regular attendance in official dance contests and championships throughout germany . the cg group consisted of 38 sedentary subjects ( 71.66 1.11 years , 30 women , ecq score : 8.43 ) with no record of dancing or sporting activities ( see section 4 for details regarding the selection of appropriate controls ) . the age distribution ( p = 0.829 ) and education level ( number of school years , p = 0.926 ) of subjects across the groups was balanced . the study was approved by the local ethics committee of the ruhr - university of bochum . during dance competitions , all subjects in the ed group were assigned to a starting group referred to as seniors iv for the competition , 10 different dances had to be performed in a mandatory order , including the slow waltz , tango , viennese waltz , slowfox , quickstep , samba , cha - cha - cha , rumba , paso doble , and jive , each of which lasted for 1.52 min . on the basis of points given by adjudicators during the contests , the subjects of our ed group were assigned the highest german grade ( s ) within the corresponding starting group . therefore , subjects of the ed group had to be particularly fit with regard to mobility , muscle flexibility , and body composition . although literature reports indicate a lower cardio - respiratory performance ( i.e. , maximal oxygen uptake or vo2max ) for professional ballet dancers in comparison to other athletes performing physical activities like running or swimming , professional modern dancers were shown to have a significantly higher maximal oxygen uptake compared to professional ballet dancers ( for review , see ) . given the average workload of 4.5 h / week plus assumed 2.5 h / week for dancing competition adds up to 7 h a week , which totals 350 h per year , which sums up to 7500 h , which is the typical workload range required to qualify for becoming expert . lifestyle and general activity levels were assessed using the everyday competence questionnaire ( ecq ) that addresses the aspects of everyday life , such as independence in activities of daily living and mobility , social relations , general health status , and life contentment . the compilation of questions used in the ecq accounts for the changing living conditions of today 's seniors . the ecq consisted of 17 items , including housekeeping , daily routines , manual skills , mobility , sports , subjective well - being , linguistic abilities , and leisure - time activities , thereby addressing instrumental activities of daily living ( iadl ) and the individual engagement in other activities of everyday life as well . these activities are not necessary for fundamental functioning , but they let an individual live independently in a community . all subjects were asked to comment on the questions with as much detail as possible , thus allowing insight into their habits and living conditions . the scores were normalized to a scale from 0 to 1 by dividing the number of points achieved by the maximum possible scores per item . general intelligence was assessed using the raven 's standard progressive matrices ( rspm ) [ 19 , 20 ] . the test was administered according to standard instructions with a 30 min time constraint . in the control group , the rspm was conducted in a pre-/post - design to provide data for a separate study . therefore , for both groups in this study , we used odd - numbered items only , resulting in a maximum score of 30 . in addition , the nonverbal geriatric concentration test ( akt ) was used to assess selective attention and concentration . for this paper - and - pencil test , subjects had to mark 20 symbols of 55 similar - looking patterns within a maximum time limit of 30 s ( figure 1 ) . the time required for each subject to complete the test sessions was averaged to evaluate individual performance . we performed multiple - choice rt measurements in a finger - selection visuotactile task adapted from the study of alegria and bertelson . an image of each hand was displayed on the monitor and 1 finger of the 10 was selected by a visual marker . subjects had to press the key corresponding to the selected finger on a hand - shaped , 10-button keyboard as fast as possible . one session consisted of 4 blocks of 100 trials each , which were separated by a short break after each block . the maximum response - to - stimulus interval for each trial was 2000 ms . we applied the romberg test , the timed up and go test , and the standing - turn test to assess each subjects ' ability to control their posture and maintain balance and to evaluate their security of gait . the romberg test is a standard neurological test addressing joint position sense ( proprioception ) and was applied in a condition with eyes either open or closed [ 23 , 26 ] . the movements of the body in relation to a perpendicular object behind the subject were monitored . the time until a subject started to lose balance was recorded ( maximal testing time was limited to 1 min ) . in the timed up and go test , subjects were asked to stand up from a sitting position , walk 3 m , return to the chair , and sit down again . the time to fulfill the task was measured . in the standing - turn test , a standing subject was asked to perform a 360-degree turn . the time and number of steps hand - arm fine - motor performance was evaluated using a computer - based test battery for clinical neuropsychological research ( mls ; dr . the system consists of a work plate with 2 pencils for left and right hand use . we tested speed , accuracy , and maintenance of upper limb position during execution of fine motor movements of the left and right arms , hands , and fingers by using the following tests for : steadiness , which evaluates the ability to achieve a prescribed arm - hand position and maintain it for a defined time period ; aiming , which evaluates the ability to accomplish fast arm - hand movements for small targets ; pin plugging , which evaluates fine and gross motor dexterity and coordination ; tapping , which evaluates the ability to perform very fast , repetitive wrist - finger movements with little emphasis on the precision of movement . touch threshold was evaluated using a staircase procedure by probing the fingertips of the left and right index fingers with von frey filaments ranging from 0.25 to 294 mn on logarithmic scaling ( marstocknervtest , marburg , germany ) . spatial 2-point discrimination thresholds ( 2pd ) were assessed on the tips of the left ( lid ) and right ( rid ) index fingers by using the method of constant stimuli [ 2729 ] . needle separations of 1.5 , 2.3 , 3.1 , 3.9 , 4.7 , 5.6 , and 7 mm were used . test - retest reliability using this procedure was 0.90 for young subjects and 0.88 for older participants . the summed responses were plotted against the needle distances resulting in a psychometric function , which was fitted using a binary logistic regression ( spss , spss inc . the threshold was taken from the fit where 50% correct responses were reached . to pool the data obtained from the various tests , we defined 5 domains covering similar functional categories . cognitive performance comprised data from the akt and the rspm . posture and balance comprised data from the romberg test , the timed up and go test , and the standing - turn test . motor performance comprised steadiness , aiming , pin plugging , and tapping . a separate domain rt was introduced to include data from the multiple - choice rt task . to compare performances across all tests and all subjects , we calculated normalized performance indices ( ips ) for each subject , and each test as ( wp - ip)/(wp - bp ) , where wp is the worst performance of all subjects , ip is the individual performance , and bp is the best performance of all subjects . indices were subsequently averaged across tasks belonging to a particular domain as described above . in all cases , we reported averages and standard error of the mean ( sem ) . we used the mann - whitney u test to detect differences between the 2 groups . moreover , we computed effect sizes according to cohen 's d . to test for differences in the distribution of ips , we used chi - square statistics . a p value of < 0.05 was considered significant . we tested cognitive , posture , balance , and sensorimotor performance in the 2 groups of older participants , matched for gender , age , and education , who had an extended history of expert and competitive dancing ( ed ) , or no dancing experience ( control group ; cg ) . the ed group had a superior performance in most of the tests ( table 1 ) . the ed group showed significantly higher ecq scores than the cg group ( z = 2.996 , p = 0.003 ) . cognitive performance assessment showed significant differences between the ed and cg groups for the rspm ( figure 2(a ) ) ( z = 2.776 , p = 0.006 ) and akt ( z = 4.997 , p 0.001 ) . for both hands , the ed group had faster rts , which were averaged for the left ( z = 2.294 , p = 0.022 ) and right hands ( z = 2.195 , p = 0.028 ) . posture and balance assessment showed significant differences between the 2 groups for the romberg test with eyes open ( z = 3.951 , p 0.001 ) , but not with eyes closed ( z = 1.250 , p = 0.211 ) . subjects in the ed group needed less time for the completion of the standing - turn test ( z = 2.815 , p = 0.005 ) . moreover , the ed group showed significantly shorter up and go times ( figure 2(b ) ) ( z = 3.819 , p 0.001 ) . significant differences were found between the 2 groups in the motor domain for the aiming subtest ( figure 2(c ) ) , which showed that there were fewer errors in the ed group ( z = 2.808 , p = 0.005 ) , as well as a shorter pin - plugging time ( z = 2.343 , p = 0.019 ) , both of which were observed in the right hand only . in the tactile domain , the assessment of 2pd thresholds showed significant differences between the 2 groups ( figure 2(d ) ) for the right ( z = 2.434 , p = 0.015 ) and left ( z = 2.515 , p = 0.012 ) index fingers . the calculation of ip for each test and each subject allowed a direct comparison of performances across all tests and all subjects and facilitated grouping into functional domains covering cognition , rts , posture and balance , motor performance , and tactile performance . as shown in table 2 , the ed group showed significantly higher ips in 2 of the 5 domains , with the largest difference being for posture and balance ( z = 5.599 , p 0.001 ) followed by rts ( z = 3.462 , p = 0.001 ) . our findings indicated a general advantage for the ed group , which spans cognitive , perceptual , and motor performance . in order to obtain insight into possible differences in the overall distribution of ips within a given domain , we grouped the ip for each domain into > 0.5 and < 0.5 values and compared the percentage of occurrence of ips > 0.5 across groups , where 0 indicates the worst and 1indicates the best performance . in 3 of the 5 domains analyzed , the cg group had a significantly higher number of ips that was lower than 0.5 ( subjects with ip < 0.5 , cognition : ed = 9.09% , cg = 36.26% , = 6.12 , p = 0.013 ; rt : ed = 4.55% , cg = 44.74% , = 12.00 , p = 0.001 ; posture and balance : ed = 21.82% , cg = 70.37% , = 25.87 , p 0.001 ) . higher ips for motor performance that were lower than 0.5 were also found within the cg group , but they did not reach statistical significance ( ed = 17.61% , cg = 24.59% , = 3.75 , p = 0.053 ) . in agreement with the results shown in table 2 , increased ips lower than 0.5 were found for tactile performance in the ed group ( ed = 38.64% , cg = 21.26% , = 5.16 , p = 0.023 ) . accordingly , the superior performance of the ed group within some domains did not come from the fact that their best performers were better than those of the cg group , but it was instead due to the fact that the ed group lacked the poor performers that were frequently present in the cg group . we have recently shown that a regular schedule of many years of amateur dancing in old age has a wide range of beneficial effects not only on posture , but also on sensorimotor and cognitive performance . this observation raised the question of whether preservation of a high level of proficiency such as that which is present in elderly expert ballroom dancers has an even higher positive impact on physical and cognitive fitness in aged individuals as compared to those with only basic amateur dancing skills . we therefore studied the impact of extended participation in competitive dancing in a group of older subjects and compared them to an aged - matched , nondancer cg . in addition to posture and balance , which are closely related to dancing , we performed a broad assessment of cognitive , attentional , perceptual , and sensorimotor abilities . according to our hypothesis about the impact of multi - year dancing activities therefore , we needed to test many different domains from cognitive functions to basic sensory abilities . criteria for selecting a test included a brief time needed to complete the test , general acceptance , and a wide extension . in this sense , a particular test served as a surrogate for a given domain , implying that other tests for this field would have shown similar effects . floor or ceiling effects have been described when using the advanced ( ceiling ) or colored progressive matrices ( floor effects ) . in our study , we used a subset of odd - numbered items only , resulting in a maximum rspm score of 30 . the scores obtained for ed ( 19.59 0.75 ) and cg ( 15.39 0.83 ) indicate a lack of floor and ceiling effects . we included a cg that was characterized by having no record of dancing or sporting activities for at least 5 years . the ecq addresses specific aspects of so - called instrumental activities of daily living , such as housekeeping , daily routine , manual skills , mobility , sports , subjective well - being , linguistic abilities , and leisure - time activities . participants in the cg had lower ecq scores , indicating a more passive and sedentary lifestyle . these data imply a close association between a lack of sporting activities and lifestyle , the identification of which was not our primary goal when we selected subjects . it is well acknowledged that selecting an adequate control group for any type of expert subpopulation poses a major challenge . for example , instead of using a group of passive individuals , one could use a group that would also be considered expert , but in a different domain . by this , the particularities of the respective areas of expertise would have been compared . as dancing is coupled with physical exercise , our controls were selected to have neither experience with dance or sports . further studies are required to test other groups comprised of individuals performing some type of sport activity in order to disentangle the effects of physical exercise hidden in dance . subjects in the ed group performed better in most of the tasks investigated in this study . however , analysis of the individual ips , which allowed comparison across all tests and subjects for the 5 domains ( cognition , rt , posture and balance , motor performance , and tactile performance ) showed a significantly better performance in the ed group with regard to rt and posture and balance only . a similar argument can be made for the finding of faster rts in the ed group , which might be attributable to the requirements for both high attention and fast and well - coordinated motor responses . in contrast to the previously studied group of elderly amateur dancers , we found no differences between the ed and cg for performance measures related to the domains of cognition and hand - arm motor functions . on the other hand , this limited generalization is , to some extent , in agreement with recent studies that concluded that although high levels of expertise may have a positive impact on functions related specifically to that proficiency , other non - expert - related abilities of older experts were similar to those of nonexpert older adults . in the previous study performed in a group of amateur dancers , we showed that the best performers of each task were present in both the dancing and the cg with similar frequency , but that the amateur dancing group lacked the number of poor performers that were frequently found within the control group . here , we showed that for non expertise - related domains such as tactile abilities , poor performers were equally present in both the ed and cg . these data led us to speculate that amateur and expert competitive dancers differ in that the latter focus on their area of expertise at the expense of other skills . since dancing at a high maintained level of expertise requires extensive practice , competitive dancing requires substantial effort with regard to the traveling time and personal strain during competitions , which might counteract the positive impact of dancing that was observed in the amateur group . another line of argument for the limited positive impact of expertise at old age comes from functional imaging studies . it has been shown that learning piano playing in amateurs elicits stronger activation in a number of brain areas in comparison to the brain activation found in professional piano players , who practice playing to maintain high levels of expertise . on the basis of these types of studies , it has been suggested that fewer cognitive resources are required for expert performance once an it is therefore conceivable that the maintenance of high - level expertise has a lesser impact than the acquisition of new skills on the general fitness of older people . for many years , dance has been successfully established as a therapeutic tool in the elderly to improve cardiovascular parameters , muscle strength , and posture and balance [ 37 , 38 ] . our motivation for investigating the beneficial effects of dancing was triggered by the hypothesis that dancing can be regarded as an equivalent of enriched environmental conditions in human individuals , because of the unique combination of physical activity , rhythmic motor coordination , emotion , affection , balance , memory , social interaction , and acoustic stimulation . however , our data suggest that many of the features that play a crucial role in promoting positive effects in amateur dancing might be less relevant for expert dancers . this assumption is supported by the observation that the positive impact of expert competitive ballroom dancing was limited to expertise - related tasks such as posture and balance , or rts . thus , years spent developing high - level expertise helps to maintain remarkable levels of posture - related performance even at an advanced age , which is in accordance with the notion that brain plasticity is operational in old age [ 27 , 39 ] . compared to activities such as exercising , walking , or playing an instrument , dance has the advantage of combining many diverse features including physical activity , social and emotional interaction , and sensory stimulation , each of which is well documented to have beneficial effects . consequently , many studies in the elderly have shown that improving aerobic capacity through physical exercise programs has beneficial effects on cognitive performance [ 6 , 8 , 9 , 4043 ] . while cardiovascular fitness might directly affect blood pressure and circulation , animal research on the effects of physical exercise suggests a crucial involvement of neurotrophins and other nerve growth factors [ 44 , 45 ] . use - dependent plasticity , synaptic efficacy , and the maintenance of synaptic connections are controlled and modulated by neurotrophins such as brain - derived neurotrophic factor ( bdnf ) . bdnf levels increase by many factors such as physical activity and social interaction [ 4447 ] . housing animals under enriched environmental conditions , in particular , has been shown to increase neurotrophin gene expression , thus exerting neuroprotective functions [ 4850 ] . mild stress response in cells has been advocated as a major driving force for the upregulation of stress resistance genes and growth factors . interestingly , among the factors inducing mild stress are sensory stimulation , physical activity , and cognitive challenges , all of which are involved in dancing . it must be recognized that the present study , as well as our previous study with amateur dancers , can not resolve the query as to whether the superior performance of either the expert or amateur dancers is due to a group preselection of particularly fit subjects tending to engage in a regular schedule of year - long dancing or due to the dancing activity per se . it is therefore conceivable that intelligent people with better balance and faster rts are those who are more likely to select dancing as a life - long avocation . recent data from an intervention study in a pre-/post - design showed that after a 6-month dance course , elderly participants improved in all tested aspects , including perception and cognition , similar to those described here . these data show that irrespective of individual predispositions , dancing activities play a crucial role in mediating a wide range of beneficial effects , depending on the dose of exercise . our data showed that a regular , multiyear schedule of expert and competitive ballroom dancing in a cohort of older individuals preserves posture and balance parameters to a remarkable extent and has a positive impact on rt . however , our results provided no evidence for more widespread beneficial effects on related domains such as tactile and cognitive performance . these findings suggest that not all doses of exercise are helpful for alleviating age - relating deterioration but hint at a more inverted u - shape , dose - response function with optimal ranges of intervention intensity required to have maximal beneficial effects . accordingly , it might be important to adjust , depending on the individual level of activity and expertise , the challenges of intervention programs to maintain health and functional independence throughout life . given the dramatic demographic changes within industrialized countries characterized by an increasing probability of reaching old and very old age , there is an urgent need for measures permitting an independent lifestyle into old age . since there is a close association between physical fitness and cognitive performance , a number of studies investigated the impact of interventional programs on the basis of dancing for the treatment of age - related functional degradation [ 37 , 38 , 53 ] . our data showed that high levels of dancing expertise can be preserved up to a very old age , thereby maintaining remarkable levels of performance in expertise - related tasks . however , tasks outside these areas of expertise showed the age - related decline typically observed in aged - matched nonexperts .
abstractdesmoid tumors ( dts ) are a group of rare and benign soft tissue tumors that result from monoclonal proliferation of well - differentiated fibroblasts . since dts tend to infiltrate and compress adjacent structures , the location of dts is one of the most crucial factors for determining the severity of the disease . furthermore , dts can further complicate the clinical course of patients when the growth is remarkably rapid , especially for dts occurring in anatomically critical compartments , including the thoracic cavity.the authors report a case of a 71-year - old man with a known mediastinal mass incidentally detected 4 months ago , presenting dyspnea with right - sided atelectasis and massive pleural effusion . imaging studies revealed a 16.4 9.4-cm fibrous mass with high glucose metabolism in the anterior mediastinum . the mass infiltrated into the chest wall and also displaced the mediastinum contralaterally . interestingly , the tumor had an extremely rapid doubling time of 31.3 days.en bloc resection of the tumor was performed as a curative as well as a diagnostic measure . histopathologic examination showed spindle cells with low cellularity and high collagen deposition in the stroma . immunohistochemical staining was positive for nuclear -catenin . based on these pathologic findings , the mass was diagnosed as dt . after surgery , there has been no evidence of recurrence of disease in the patient.this patient presents a mediastinal dt with extremely rapid growth . notably , the doubling time of dt in our case was the shortest among reported cases of dt . our experience also highlights the benefits of early interventional strategy , especially for rapidly growing dts in the thoracic cavity .	desmoid tumors ( dts ) , also known as aggressive ( deep ) fibromatosis or musculoaponeurotic fibromatosis , are a heterogeneous group of rare and benign soft tissue tumors caused by monoclonal proliferation of well - differentiated fibroblasts . these tumors are rare entities that account for only 0.03% of neoplasms and less than 3% of soft - tissue tumors . previous studies have reported that the estimated annual incidence of dts is 1 to 4 per million population . although dts do not metastasize , they tend to infiltrate and/or compress adjacent organs , manifesting diverse clinical symptoms . thus , the location is an important factor that determines the clinical course of dts . several therapeutic strategies for dts have been suggested , including close observation , hormonal therapy , radiation , and curative surgery . specifically , given that the incidence of dts in the thoracic cavity is uncommon and that mediastinal dts are quite rare with only approximately 20 patients reported in the english literature , critical factors that clinicians should take into account when they decide the treatment strategy for mediastinal dts are not well understood . herein , we describe a case of a rapidly growing dt in the anterior mediastinum with a calculated doubling time of 31.3 days , the shortest among reported cases of dts . the rapid growth of dt complicated the clinical course of the patient , causing massive pleural effusion and atelectasis , chest wall invasion , mediastinal shifting , and compression of the diaphragm . the mediastinal dt was completely removed through surgery , and there has been no evidence of recurrence of the disease in the patient . a 71-year - old man visited our emergency department because of 1-week history of dyspnea with productive cough and right chest pain that aggravated with daily activities . he had undergone aortic repair because of aortic valve regurgitation caused by aneurysm of the aortic root and ascending aorta 2 years before this presentation . four months before admission , a regular check - up none - enhanced chest computed tomography ( ct ) scan had shown a 7.4 3.4-cm mass in the anterior mediastinum incidentally but no further evaluation was made because of patient refusal ( fig . ( a ) initial axial image of chest ct ( none - enhanced ) taken at a local clinic displays a tumor ( arrows ) in the anterior mediastinum . ( b ) axial image of chest ct ( contrast - enhanced ) taken at admission to our clinic 4 months after initial detection of the mass demonstrates extremely rapid growth of the anterior mediastinal tumor ( arrows ) . the tumor mainly consists of soft tissue matter with a radiodensity of 27 hu on none - enhanced ct . when iodine - based contrast medium was administered , the tumor was enhanced to only 33 hu , implying hypovascularity . ( c ) pre - contrast ; ( d ) arterial ; ( e ) portal ; ( f ) delayed - phase dynamic gadolinium - enhanced chest magnetic resonance imaging ( t1-weighted image ) reveals a delayed enhancement pattern , indicating a fibrous tumor . ( g ) coronal image of gadolinium - enhanced magnetic resonance imaging ( t2-weighted image ) shows infiltration of the tumor into the chest wall ( arrows ) and displacement of the right diaphragm to the abdominal cavity by the tumor . ( h ) 2-deoxy-2-[18f]-fluoro - d - glucose positron emission tomography - ct displays elevated glucose metabolism ( maximum standardized uptake value = 5.68 ) . his breathing was shallow , and breathing rate was slightly increased ( respiratory rate = 25/min ) , but other vital signs were within the normal range . laboratory data revealed that platelets , white blood cells , and differential cell counts were all within the normal range . arterial blood gas analysis exhibited respiratory alkalosis with metabolic compensation , and arterial oxygen saturation was 96% on pulse oximetry in ambient air . on physical examination , there were decreased breath sounds on the right lower chest , and percussion revealed dullness over that area . subsequent pleural fluid examination demonstrated neutrophil - dominant ( 68% ) exudate according to light criteria [ pleural fluid protein [ 4.8 g / dl ] to total serum protein ( 6.4 g / dl ) ratio was 0.75 ] . subsequent ct scan displayed a 16.4 9.4-cm tumor in the anterior mediastinum ( fig . 1b ) . the tumor had a homogeneous attenuation of 27 hounsfield units ( hu ) on nonenhanced ct , and the tumor had 33 hu after enhancement , indicating that the tumor possessed dominantly soft tissue components with hypovascularity . a shift of the mediastinum to the opposite site of the mass and compression of the right - side heart were also evident ( fig . 1b ) , but there was no evidence of hemodynamic compromise in the patient on echocardiography . moreover , passive atelectasis was visible in the right upper and middle lobes . there were no enlarged lymph nodes in the thoracic cavity . because the most frequently occurring masses in anterior mediastinum are thymoma , lymphoma , and teratoma , the radiologic features of the mass on ct scan of our case were very unusual and atypical . in fact , thymoma and lymphoma have been reported to have mild to moderate enhancement , and teratoma often exhibits heterogeneous contents of fat , fluid , and soft tissue , as well as calcification . to further characterize the mass and to evaluate whether the mass was involved in the major vascular system , chest magnetic resonance imaging ( mri ) the mass showed high signal intensity on t2-weighted images and low signal intensity on t1-weighted images . in addition , the dynamic contrast - enhanced mri exhibited inhomogeneous enhancement in portal and delayed phases , suggesting that the mass can be a fibrous tumor ( fig . 1c to f ) . t2-weighted mri also demonstrated tumor infiltration into the left chest wall at the fifth to seventh rib locations and displacement of the right diaphragm to the abdominal cavity by the tumor ( fig . interestingly , the calculated volume of the tumor by the simple sum - of - area technique , revealed that the tumor had increased from 302.5 11.68 cm to 4103.4 158.4 cm over a period of 116 days ( an approximately 13-fold increase in volume ) . according to the formula to estimate doubling time , moreover , positron emission tomography showed uneven avid glucose uptake ( maximum standardized uptake value = 5.68 ) ( fig . 1h ) . for the pathologic diagnosis of the mass , percutaneous transthoracic needle biopsy ( ptnb ) was performed . the result , however , was inconclusive because of scant cellular components of the specimen . thus , we planned surgical resection of the tumor for a curative as well as a diagnostic purpose . the largest diameter of the tumor was approximately 20 cm in the operation field . 2a ) such as the fifth to seventh ribs and the lower sternum , the pericardial space , and the medial portion of the diaphragm ( fig . en bloc resection of the mass consisted of partial resections of the fifth and the sixth ribs , the intercostal muscles , the pericardium , and the diaphragm ( fig . the mass was successfully resected , and histopathologic examination of the specimen revealed spindle cells with low cellularity and high collagen deposition in the stroma without features of malignant cells , such as nuclear hyperchromatia , nuclear atypia , high nuclear - to - cytoplasmic ratio , and mitosis ( fig . the patient recovered after surgery without any complications , and there has been no evidence of recurrence of dt so far . ( a ) severe adhesion ( arrows ) between the tumor and the adjacent chest wall ( asterisk ) is present in the operation field . ( b ) the tumor shows severe adhesion ( arrows ) to the medial portion of the diaphragm . ( c ) the size of the tumor specimen is 22 20 11.3 cm . surgical resection of the mass includes a part of the ribs and the intercostal muscles ( arrow ) , the diaphragm ( open arrow ) , and the pericardium ( arrow head ) . ( d ) cross - section shows gelatinous appearance and a heterogeneous mixture of white and light green color of the tumor . ( e ) hematoxylin & eosin staining of the specimen demonstrates a relatively small number of spindle cells in a collagen - abundant stroma ( 400 ) . there are no features of malignant cells , such as nuclear hyperchromatia , nuclear atypia , high nuclear - to - cytoplasmic ratio , and mitosis . ( f ) immunohistochemical staining with -catenin demonstrates strong positive reactions in the nuclei of these cells ( 400 ) . merged color of blue and red represents positivity for nuclear -catenin staining . in this case , the patient presented an extremely rapid growing anterior mediastinal mass that had grown approximately 13-fold in volume within 116 days . the local effects of this rapid growing mass , such as chest wall invasion , mediastinal shifting , and compression of the heart , induced pleural effusion and atelectasis resulting in dyspnea , cough , sputum , and right chest pain . interestingly , the growth of dt in our patient was extraordinarily rapid compared to previously reported patients of dts in the literature . previous studies have demonstrated that the location of dts is one of the crucial factors for determining clinical course . in particular , for example , several patients of mediastinal dts have shown to display severe illnesses , including cardiac arrhythmia , circulatory collapse secondary to superior vena cava syndrome , and cardiac failure . in our patient , compression of the right heart by an anterior mediastinal dt was evident without hemodynamic collapse . other clinical manifestations associated with the location of dt in the thoracic cavity of limited space were massive pleural effusion , atelectasis , chest wall infiltration , and various mass effects , including mediastinal shifting and the compression of the diaphragm resulting in severe dyspnea , cough with sputum , and right chest pain . hence , our experience also highlights the significance of anatomic location as one of the key factors for determining the severity of mediastinal dts . a previous report illustrated a dot - plot graph presenting the size of an anterior mediastinal dt over time in a patient ; the maximal doubling time was more than 9 months . in case of abdominal dts , a previous case report showed a dt with a doubling time of 122 days , and another study of 23 patients with abdominal dts reported that the mass doubled in less than 3 month at most . in our patient , the volume of the tumor initially detected in the local clinic was approximately 300 cm . because further evaluation was not made at that time , the mediastinal dt increased in volume to approximately 4000 cm . notably , the doubling time of dt in our patient was the shortest among reported cases of dts . furthermore , the doubling time in this patient is noteworthy compared with several malignant tumors ( pancreatic carcinoma , 18255 days ; nonsmall cell lung carcinoma , 81092 days ; and small cell lung carcinoma , 54132 days ) . taken together , the rapid growth of the dt in our patient may have played a key role in the complicated clinical course . based on our experience , masses with rapid volumetric growth may cause serious complications in the thoracic cavity , a representative of closed anatomic compartments . thus , an early invasive therapeutic strategy is likely to be beneficial for rapidly growing mediastinal dts . obtaining tissue specimens through minimally invasive procedures , such as ptnb , enables clinicians to diagnose the disease promptly and allow patients to recover from procedure - associated morbidity early , thereby possibly yielding favorable outcomes in certain clinical situations . as for dts , however , the role of ptnb in preoperative diagnosis is yet to be defined . in our patient , the result of ptnb was inconclusive because of a small number of cells in the specimen , and the mass was finally diagnosed as dt after surgical resection . in addition , the mass possessed characteristic findings of fibrous - dominant tumor with minimal contrast enhancement on preoperative radiologic images . likewise , there were abundant fibrous contents with low cellularity in pathologic specimens , which makes the pathologic diagnosis somewhat inconclusive despite small biopsy such as ptnb . based on our experience , early surgical resection rather than small biopsies might be a reliable diagnostic method , especially for soft tissue tumors having fibrous - dominant components on radiologic images . in this report , we described an interesting case of a rapidly growing dt in the anterior mediastinum . the aggressive nature of the dt in our patient was highlighted by calculating the doubling time that indicated extremely rapid growth . considering that dts commonly infiltrate and compress adjacent organs , its deleterious effects can be further intensified by rapid growth in the mediastinum . our experience highlights dt as one of the differential diagnoses for rapidly growing mediastinal tumors and the benefits of early intervention of rapidly growing mediastinal dt . the institutional review board of chonbuk national university hospital stated that it was not necessary to achieve irb approval for this case report , but that patient consent was required as the study dealt only with retrospective use of the patient 's medical records and related images . written informed consent was obtained from the patient before the publication of this case report and accompanying images .
the exact cellular and molecular mechanisms of sepsis - induced encephalopathy remain elusive . the breakdown of the blood - brain barrier ( bbb ) is considered a focal point in the development of sepsis - induced brain damage . contributing factors for the compromise of the bbb include cytokines and chemokines , activation of the complement cascade , phagocyte - derived toxic mediators , and bacterial products . to date , we are far from fully understanding the neuropathology that develops as a secondary remote organ injury as a consequence of sepsis . however , recent studies suggest that bacterial proteins may readily cross the functional bbb and trigger an inflammatory response in the subarachnoid space , in absence of a bacterial invasion . a better understanding of the pathophysiological events leading to septic encephalopathy appears crucial to advance the clinical care for this vulnerable patient population .	the clinical study by van der boogaard and colleagues recently published in critical care was designed to unravel some of the open questions regarding the pathophysiology of septic encephalopathy . the authors mimicked inflammation - associated encephalopathy by induction of experimental endotoxemia using escheria coli - derived lipopolysaccharides ( lpss ) in 15 healthy young volunteers . outcome parameters were serum levels of cytokines , cortisol , neuron specific enolase , s100- , as well as electroencephalographic changes and cognitive function in comparison to a healthy cohort of ten control volunteers . interestingly , van der boogaard and colleagues described that the endotoxin - induced ' cytokine storm ' and cortisol release failed to provoke any signs of septic encephalopathy . no clinically relevant electroencephalographic changes occurred , and markers of neuronal damage ( neuron specific enolase , s100- ) were found to be slightly reduced following lps challenge . endotoxemia even resulted in a higher state of alertness and improved cognitive function in comparison to the healthy cohort . the authors concluded that temporary systemic inflammation caused by endotoxemia can not provoke the development of septic encephalopathy . nonetheless , their present study shed some further light towards our understanding of the immunological pathophysiology of septic encephalopathy , as it appears unlikely that bacterial lps is a driving force in the development of septic encephalopathy . note worthy , the spectrum of responsible microorganisms has shifted from predominantly gram - negative bacteria in the late 1970s and 1980s to predominantly gram - positive bacteria and fungal infections at present . the authors ' findings underscore the complexity and ambiguity of septic encephalopathy , which continues to be a puzzling complication of the sepsis syndrome . this is of particular concern , as up to 70% of all septic patients develop signs of such brain damage . traditionally , septic encephalopathy was thought to occur due to inflammatory breakdown of the blood - brain barrier ( bbb ) as a ' key ' causative factor of sepsis - associated delirium . a dysfunction of the bbb has been shown to be induced by various inflammatory mediators , such as il-1 , tnf- , complement , and bradykinin , which can cause a ' sterile meningitis ' in the absence of a bacterial pathogen . moreover , complement c3 and c5a have been linked to sepsis - induced compromise of the bbb . of note , direct contact between blood and cerebrospinal fluid leads to complement activation , as may be the case in severe bbb dysfunction . the disruption of this physical barrier then allows circulating neurotoxic substances to extravasate into the brain parenchyma and promote an inflammatory response . however , this traditional notion of initial bbb compromise prior to development of septic encephalopathy has recently been challenged . in their experimental study , londoo and cadavid injected mice intraperitoneally with labeled outer membrane lipoproteins of borellia turicatae and monitored their localization in the brain . surprisingly , two of the lipoproteins studied ( lvsp1 and lvsp2 ) were capable of disseminating from the periphery into the brain and caused intracerebral inflammation without intracerebral spirochete accumulation . another piece of the complex puzzle of septic encephalopathy may be the extensive communication between the nervous and the immune system . interestingly , this interaction is bi - directional , as cytokines can trigger the release of glucocorticoids via the hypothalamic - pituitary axis , and , in turn , glucocorticoids suppress cytokine synthesis of leukocytes . moreover , both systems use a common biochemical language of hormones , ligands and receptors to communicate with each other . in the setting of sepsis , the majority of work in neuroimmunology has focused on the anti - inflammatory properties of the vagus nerve , popularized by the term ' the inflammatory reflex ' . while these interactions are likely to be involved in the development of septic encephalopathy , the exact mechanisms remain inadequately understood . one of the dilemmas in current sepsis research is the poor transferability of promising experimental findings . many pharmacological research strategies have failed a successful translation from ' bench to bedside ' . this predicament is likely caused by an obvious disconnect between controlled animal models and the heterogeneous clinical sepsis syndrome observed in humans . experimental human studies , such as the study by van der boogaard and colleagues , are limited by several factors . endotoxemia is usually induced in a young , healthy population , and may rather present an acute intoxication model than the multi - microbial or fungal infections observed in the sepsis syndrome . in such an experimental setting , the timing and dosage of lps has to be limited based on safety issues , and therefore might not reach the threshold for the development of a significant bbb damage . moreover , sepsis results from various causative etiologies , and susceptibility is influenced by premorbid factors , including ethnicity , gender , age , genetic defects and environmental factors . the belief that a single key mediator causes sepsis , and that its neutralization could be a cure for all patients with sepsis , seems erroneous . in particular , pre - existing genetic and epigenetic changes , mutations in genes that encode pattern - recognition receptors or inflammatory mediators , may have an enormous impact on the host 's susceptibility to sepsis . interdisciplinary approaches involving both clinicians and basic scientists will be necessary to improve our knowledge of the underlying pathophysiology of sepsis and septic encephalopathy . such interdisciplinary , large - scale programs involving surgery , genomics , proteomics , biostatistics , bioinformatics , computational biology and genetics are currently underway . bbb : blood - brain barrier ; il : interleukin ; lps : lipopolysaccharide ; tnf : tumor necrosis factor .
orthodontics from time immemorial has always been considered as the first speciality of dentistry . diagnosis and treatment planning from dr . tweed 's time always required extraction of four vital and functional premolar teeth . however , many patients , parents and general dentists have failed to understand that orthodontic treatment at an early age can help save the extraction teeth to achieve a full functional and esthetic harmonius dentition . our case demonstrates a non - extraction orthodontic treatment plan of a developing skeletal class ii malocclusion . the results of this case show a successful correction of class ii molar malocclusion with excellent form and function , which resulted in improvement of the patient 's frontal and facial profile , which , thereby boosted her personality and self - confidence .	hence , the diagnosis arrived at was to treat it with very affordable activator therapy followed by inexpensive fixed appliance brackets . habits play a significant role in improper development of dental as well as facial form , function and esthetics . moreover , it is known from child psychology that habits are many a times correlated with stress or anxiety in a growing child . of the very many common childhood habits , it results in compromised dental and facial esthetics by posterior positioning of the mandible , protrusion of maxillary anterior teeth and deep bite . such children on reaching maturity get conscious of these profile problems leading to a loss of self - confidence , which could even hamper their social development . a 10 years 6 months - old girl , born on 06/02/1998 , presented in the department of dentistry , bhatia general hospital , under parental guidance with a complaint of protruding upper front teeth . her medical history was non - contributory . according to the history charting questionnaire , her mother revealed her daughter 's lip biting habit while studying or reading . the habit had developed since childhood and , even after repeated counselling , efforts taken to quit the habit by her previous general practitioner proved in vain . her extraoral examination [ figures 12 ] revealed a shortened lower facial height with a convex profile . a clinically flat mandibular plane was also noted , signifying a horizontal growth pattern , favorable for activator therapy . the face was symmetrical and the nasolabial angle was approximately 90.on temporomandibular joint ( tmj ) examination , all movements were found to be normal . her visual treatment objective ( vto ) clinically showed a marked improvement in profile guaranteeing a successful result . pre - treatment extraoral frontal view pre - treatment extraoral lateral profile view on intraoral examination [ figures 35 ] , the molar / canine relation was end - on tending toward angle 's class ii relation with a partially impacted lower left canine . pre - treatment intraoral view at occlusion pre - treatment intraoral left lateral view at occlusion pre - treatment intraoral right lateral view at occlusion cephalometric analysis showed the sella - nasion - point a angle ( sna ) to be 82.4 and sella - nasion - point b angle ( snb ) to be 76.1. the resulting point a - nasion - point b angle ( anb ) of + 6.3 suggested a posterior placement of the mandible . upper incisor to the sella - nasion ( sn ) plane was 120and lower incisor to mandibular plane ( impa ) was 110 , reconfirming upper and lower incisor proclination . frankfort 's mandibular plane angle ( fma ) of 17 suggested a horizontal growth pattern , which was very favorable for the use of a myofunctional appliance . the orthopantomogram showed continuous posterior and lower borders , normally positioned condyle , a pre - requisite for myofunctional treatment , adequate alveolar bone height and good root parallelism with a partially impacted lower left canine . the ashley howe 's model analysis turned out to be 44% , revealing a borderline case for an extraction decision . thus , the patient was diagnosed as a case of developing skeletal class ii malocclusion , exhibiting an end - on molar and canine relation . andreasen 's modified activator with a lower lip pad was the appliance of choice for treating the class ii skeletal base malocclusion . this was finished with pre - adjusted fixed appliances using mclaughlin , bennett and trivisi 's 0.022 slot prescription . on post - myofunctional therapy [ figures 6 and 7 ] , the facial profile straightened and the lower one - third facial height increased significantly . post - treatment extraoral frontal view post - treatment extraoral lateral profile view intraorally [ figures 810 ] , the molar / canine relation turned into angle 's class i relation bilaterally . post - treatment intraoral frontal view at occlusion post - treatment intraoral left lateral view at occlusion post - treatment intraoral right lateral view at occlusion cephalometrically , sna stayed constant whereas snb increased to 79.6,which resulted in an anb of + 2.8 , normal for our indian population . both upper incisor to sn plane and impa reduced dramatically , resulting in an interincisal angle of 128 , corroborating a decrease in the upper / lower incisor proclination . fma increased to 22 , also adding to the near - normal vertical component of growth . these skeletal / dentoalveolar changes could be attributed to ( i ) correction of the lower lip biting habit , ( ii ) remodelling at the tmj , ( iii ) restriction of the maxillary growth and ( iv ) inherent increase in the natural mandibular bone mass . dentoalveolar changes included angle 's class i relation , which was the end result of a correct bite registration procedure . habit correction included keeping the lower lip away by means of the lower lip pad , which resulted in pacification of the hyperactive mentalis muscle activity . registration of bite was an important feature as angle 's class i occlusal relation , which was obtained after following the correct bite registration procedure . although bite registration requires case - specific parameters , a posterior clearance of 3.5 mm , an anterior clearance of 2.5 mm and guidance into angle 's class i occlusal relation were followed . developing skeletal class ii malocclusion can be most effectively treated by non - extraction means if diagnosed at an early age and with correct patient parent motivation . as witzig has rightly said , there are no bad patients or appliance that fail , it 's we doctors who fail to motivate the patient for a correct patient - appliance combination .
backgroundadequate hemostasis in cranial and spinal tumor surgery is of paramount importance in neurosurgical practice . generalized ooze bleeding from the surgical walls cavity , coming from neoplastic vessels or nervous tissue , may be problematic . recent technical advances have dramatically reduced intraoperative complications related to blood loss . several techniques are usually employed to control hemostasis in tumor surgery , including preoperative embolization , intraoperative hypotension , electrical coagulation , and local application of fibrin sealants or hemostatic matrix , which influence coagulation.material/methodsour aim in this study was to evaluate the efficacy and the safety of the aquamantys system ( medtronic advanced energy , portsmouth , nh , usa ) , a novel bipolar coagulation device that incorporates a new bipolar coagulation technique . this device has been used in 10 consecutive patients affected by cerebral tumor along with the standard microsurgical technique and well - known intraoperative tools . the technique is associated with simultaneous delivery of bipolar radio frequency energy and conductive fluid through its electrode tip . the volume of saline passing by the electrode tip prevents charring and maintains a clean tip . this cools the tissue as it raises the temperature sufficiently to shrink the collagen of veins and arteries.resultseffective hemostasis was achieved in all the cases . no complications or unwanted reactions associated with the device have been observed.conclusionsour findings suggest that the aquamantys system may be a highly effective adjuvant tool in minimizing blood loss in a patient with brain tumor , as well as reducing time of surgery .	several strategies are used to reduce blood loss , including preoperative embolization , intraoperative controlled hypotension , electrical coagulation , or local application of fibrin sealants or hemostatic matrix , which influence coagulation [ 13 ] . with usual contact methods involving electrocautery , such as monopolar knife and bipolar coagulator , removal of the probe from the adherent , desiccated eschar around the probe often detaches the eschar , causing greater bleeding than before the coagulation . in this scenario , the aquamantys system ( medtronic advanced energy , portsmouth , nh , usa ) incorporates a new bipolar coagulation technique associated with simultaneous delivery of bipolar radio frequency energy and conductive fluid through its electrode tip . the volume of saline passing by the electrode tip prevents charring and maintains a clean tip . this cools the tissue as it raises the temperature sufficiently to shrink the collagen of veins and arteries . power and saline flow are adjustable for different types of tissue to promote effective coagulation . we report a series of brain tumor patients undergoing surgery using the aquamantys system , along with its epidural vein sealer ( evs ) , which revealed some practical advantages . the aquamantys system ( medtronic advanced energy , portsmouth , nh , usa ) is based on a new bipolar coagulation technique , the so - called transcollation technology , a proprietary combination of radiofrequency energy and saline . the technique is associated with simultaneous delivery of bipolar radio frequency energy and conductive fluid through its electrode tip . briefly , the system works by combining a bipolar electrosurgical generator with a rotary peristaltic pump to provide simultaneous delivery of radiofrequency energy and saline when used with aquamantys handheld disposable devices . the saline cools the tissue as it is treated and evenly conducts the energy into the tissue to seal blood vessels . the thermal effect shrinks the collagen in the vessel walls of small arteries , and this results in the cessation of bleeding from these vessels . the saline used as a conductive fluid cools the tissue surface and prevents the surface temperature from getting too hot , thus avoiding charring . the aquamantys device , when associated with the handpiece evs , has a shaft diameter of 4 mm , thus offering good visibility and less crowdedness in the surgical field ( figure 1 ) . the aquamantys system has been used in 10 consecutive patients with brain tumors ( 5 meningiomas , 3 glioblastomas , and 2 pituitary adenomas ) . in cases with pituitary adenomas , the device was used for hemostasis of the bleeding from the nasal mucosa and on the tumor capsule . the coagulation setting , adjustable from 20 to 200 watts , was 20 watts and the saline flow ( with settings for low , medium , and high ) , was set in medium the device was used in the same way as standard electrocautery except that the bipolar sealer could be used not only in a localized manner to stop bleeding , but also in a spreading manner to stop diffuse bleeding . the aquamantys system ( medtronic advanced energy , portsmouth , nh , usa ) is based on a new bipolar coagulation technique , the so - called transcollation technology , a proprietary combination of radiofrequency energy and saline . the technique is associated with simultaneous delivery of bipolar radio frequency energy and conductive fluid through its electrode tip . briefly , the system works by combining a bipolar electrosurgical generator with a rotary peristaltic pump to provide simultaneous delivery of radiofrequency energy and saline when used with aquamantys handheld disposable devices . the saline cools the tissue as it is treated and evenly conducts the energy into the tissue to seal blood vessels . the thermal effect shrinks the collagen in the vessel walls of small arteries , and this results in the cessation of bleeding from these vessels . the saline used as a conductive fluid cools the tissue surface and prevents the surface temperature from getting too hot , thus avoiding charring . the aquamantys device , when associated with the handpiece evs , has a shaft diameter of 4 mm , thus offering good visibility and less crowdedness in the surgical field ( figure 1 ) . the aquamantys system has been used in 10 consecutive patients with brain tumors ( 5 meningiomas , 3 glioblastomas , and 2 pituitary adenomas ) . in cases with pituitary adenomas , the device was used for hemostasis of the bleeding from the nasal mucosa and on the tumor capsule . the coagulation setting , adjustable from 20 to 200 watts , was 20 watts and the saline flow ( with settings for low , medium , and high ) , was set in medium the device was used in the same way as standard electrocautery except that the bipolar sealer could be used not only in a localized manner to stop bleeding , but also in a spreading manner to stop diffuse bleeding . no complications or unwanted reactions associated with the device were observed . on the removal of meningioma , at first this technique not only coagulated superficial blood vessels in the tumour capsule , but also produced shrinkage of the capsule due to the heating effect , facilitating the dissection of the tumor from the surrounding arachnoid planes with minimal manipulation of surrounding structures . the aquamantys probe can be used with both the naked eye and under the surgical microscope . a thin and a relative flexible probe ( tip diameter of 3.4 mm , distal bayonet shaft length of 139.45 mm ) particularly increased the usefulness of the system for hemostasis under the microscope . thus , the total amount of bleeding was reduced and the reduction of the tumor size due to shrinking made it very easy to handle the tumors . in glioma surgery , the device , with its relatively wide handpiece tip , allowed optimal tumor coagulation during dissection and stopped generalized ooze bleeding from the surrounding surgical wall cavity . in brain tumors that have a large blood supply from feeding arteries penetrating the skull base , the aquamantys device was extremely useful for the hemostasis of these bone - penetrating feeding arteries . the aquamantys device was also useful for troublesome bleeding from the nasal mucosa with the endonasal approach for a pituitary adenoma . by using its long distal bayonet , it was possible to stop the bleeding from the sidewall of the nasal mucosa . furthermore , the aquamantys device was very useful in coagulating the tumor capsule , leading to lesion shrinking . neurological examination on admission showed mild left hearing impairment and mild gait disturbance with ataxia . no other neurological findings ( e.g. , facial dysesthesia or facial palsy ) were observed . the patient underwent brain computed tomography ( ct ) , which showed a lesion in the left cerebellopontine angle . the mass was approximately 6 cm in diameter , appearing hypointense on t1-weighted imaging , and heterogeneously intense on t2-weighted imaging . the lesion had peritumoral edema apparent in the surrounding cerebellum on fluid - attenuated inversion recovery imaging . the tumor margins were clear and no contact with the internal auditory meatus was evident . slight dural tail sign was evident on the left sigmoid sinus , which was infiltrated but not occluded by the lesion ( figure 2 ) . left suboccipital retrosigmoid craniotomy exposed the tumor originating from the tentorium , and present mainly in the infratentorial epiarachnoid space , with a small attachment to the edge of the petrosal bone . the lesion was well demarcated from the surrounding cerebellum , but attachment to the petrosal vein was identified . the aquamantys device was used both for tumor capsule coagulation and meningioma shrinking ( figure 3 ) . bipolar coagulation and ultrasound suction were used to achieve tumor resection . a small piece of tumor was left inside the sigmoid sinus . two years later , brain mri follow - up did not show development of the residual tumor inside the left sigmoid sinus ( figure 4 ) . on the removal of meningioma , at first , the tumor capsule was coagulated by the aquamantys device . this technique not only coagulated superficial blood vessels in the tumour capsule , but also produced shrinkage of the capsule due to the heating effect , facilitating the dissection of the tumor from the surrounding arachnoid planes with minimal manipulation of surrounding structures . the aquamantys probe can be used with both the naked eye and under the surgical microscope . a thin and a relative flexible probe ( tip diameter of 3.4 mm , distal bayonet shaft length of 139.45 mm ) particularly increased the usefulness of the system for hemostasis under the microscope . thus , the total amount of bleeding was reduced and the reduction of the tumor size due to shrinking made it very easy to handle the tumors . in glioma surgery , the device , with its relatively wide handpiece tip , allowed optimal tumor coagulation during dissection and stopped generalized ooze bleeding from the surrounding surgical wall cavity . in brain tumors that have a large blood supply from feeding arteries penetrating the skull base , the aquamantys device was extremely useful for the hemostasis of these bone - penetrating feeding arteries . the aquamantys device was also useful for troublesome bleeding from the nasal mucosa with the endonasal approach for a pituitary adenoma . by using its long distal bayonet , it was possible to stop the bleeding from the sidewall of the nasal mucosa . furthermore , the aquamantys device was very useful in coagulating the tumor capsule , leading to lesion shrinking . neurological examination on admission showed mild left hearing impairment and mild gait disturbance with ataxia . no other neurological findings ( e.g. , facial dysesthesia or facial palsy ) were observed . the patient underwent brain computed tomography ( ct ) , which showed a lesion in the left cerebellopontine angle . the mass was approximately 6 cm in diameter , appearing hypointense on t1-weighted imaging , and heterogeneously intense on t2-weighted imaging . the lesion had peritumoral edema apparent in the surrounding cerebellum on fluid - attenuated inversion recovery imaging . the tumor margins were clear and no contact with the internal auditory meatus was evident . slight dural tail sign was evident on the left sigmoid sinus , which was infiltrated but not occluded by the lesion ( figure 2 ) . left suboccipital retrosigmoid craniotomy exposed the tumor originating from the tentorium , and present mainly in the infratentorial epiarachnoid space , with a small attachment to the edge of the petrosal bone . the lesion was well demarcated from the surrounding cerebellum , but attachment to the petrosal vein was identified . the aquamantys device was used both for tumor capsule coagulation and meningioma shrinking ( figure 3 ) . bipolar coagulation and ultrasound suction were used to achieve tumor resection . a small piece of tumor was left inside the sigmoid sinus . two years later , brain mri follow - up did not show development of the residual tumor inside the left sigmoid sinus ( figure 4 ) . the development of bipolar coagulation began with the broad thermal - dispersion electrodes and has evolved to include highly accurate devices that deliver thermal energy via nonstick methods . over time , the operative range of coagulation instruments has dramatically increased and improved . however , most of the contact methods of electrocautery have 2 shortcomings . coagulation in a bloody field only leads to boiling and then carbonization of the blood . second , the coagulation of the pooled blood and not the bleeding vessel leads to the formation of a coating eschar around the probe . removal of the probe from the adherent , desiccated eschar often detaches the eschar , causing greater bleeding than before the coagulation . in these contexts , a new electrosurgery modality , the aquamantys system , which has been used successfully in orthopedic , general and cardiac surgery , is gaining interest as an innovative method . the aquamantys system and bipolar sealers feature innovative transcollation technology , a combination of radiofrequency ( rf ) energy and saline that provides hemostatic sealing of soft tissue and bone during surgery . the generator delivers transcollation technology , simultaneous rf power and saline delivery , to the disposable bipolar sealers . power settings can vary from 20 to 200 watts and 3 different flow rates can be selected . the 6.0 bipolar sealer is mainly used in orthopedic surgery , where the system has been demonstrated to be useful in reducing blood loss during hip and knee replacement procedures and trauma surgery . the evs , with its out - of - body shaft length of 172 mm and shaft diameter of 4 mm , is recently used during spine surgery such as decompression procedures ( e.g. , discectomy and laminectomy ) and all procedures requiring access to the epidural space . in our surgical procedures , we used the evs handpiece . the coagulation setting was 20 watts and saline flow was set at medium . compared with other high - frequency contact coagulation methods , the depth of necrosis , due to thermal injury , is extremely superficial and the continuous saline irrigation reduces the temperature in and around the treated area , which is crucially advantageous for neurosurgical operations . the use of standard electrocautery results in a high tissue surface temperature and therefore leads to charring of the tissue and scar formation . preventing this charring and searing of the tissue by using the aquamantys device should avoid the possible postoperative breakage or detachment of scar and clots produced by conventional electrocautery , which can lead to postoperative blood loss . since the aquamantys system uses a round handpiece tip , the tissue surface that can be treated is larger than that usually coagulated by the classical bipolar forceps . this feature allows better control of capillary ooze , a well - known problem at the end of intracerebral tumor removal , and a wide coagulation area of the tumor and its capsule . in meningioma surgery , capsule coagulation leads to tumor devascularization ; by increasing the power of the transcollation technology , tumor shrinking can be obtained . furthermore , in skull base meningioma , when simpson grade i can not be achieved , the aquamantys tip device allows an efficacious coagulation of dural attachment . aquamantys device can be applied in a brushwork fashion over large areas and it is particularly well - suited for treating diffuse bleeding and broad tumors , even when it is difficult to determine the bleeding point . furthermore , since the device does not cause carbonization or vaporization and hence does not generate smoke , the surgeon s operative view is not disturbed . the disadvantages of aquamantys should also be considered . in some instances , when the bleeding comes from a well - recognized broken vein or artery , the bipolar forceps are more adequate and precise in controlling the bleeding than is the aquamantys system . furthermore , the limited flexibility of the handpiece is a drawback , especially in case of a deep , narrow , and irregular operating channel . in such a case , however , the bipolar coagulation needs a wide enlargement of the working channel to provide complete control of the bleeding . the aquamantys system is a novel coagulation technique with an innovative transcollation technology , a combination of radiofrequency ( rf ) energy and saline that provides hemostatic sealing of soft tissue and bone during surgery . this technology can be considered as an adjuvant strategy for achieving hemostasis in brain tumor surgery . the aquamantys device is complementary to rather than competitive with bipolar coagulation and a conventional monopolar knife . the use of this device is certainly no substitute for a good microsurgical hemostasis technique . with the development of tailored and more flexible probes , the device might become a valuable surgical device .
objective . the anti - inflammatory proteins annexin - a1 and galectin-1 have been associated with tumor progression . this scenario prompted us to investigate the relationship between the gene and protein expression of annexin - a1 ( anxa1/anxa1 ) and galectin-1 ( lgals1/gal-1 ) in an inflammatory gastric lesion as chronic gastritis ( cg ) and gastric adenocarcinoma ( ga ) and its association with h. pylori infection . methods . we analyzed 40 samples of cg , 20 of ga , and 10 of normal mucosa ( c ) by the quantitative real - time pcr ( qpcr ) technique and the immunohistochemistry assay . results . high anxa1 mrna expression levels were observed in 90% ( 36/40 ) of cg cases ( mean relative quantification rq = 4.26 2.03 ) and in 80% ( 16/20 ) of ga cases ( mean rq = 4.38 4.77 ) . however , lgals1 mrna levels were high ( mean rq = 2.44 3.26 ) in 60% ( 12/20 ) of the ga cases , while low expression was found in cg ( mean rq = 0.43 3.13 ; p < 0.01 ) . normal mucosa showed modest immunoreactivity in stroma but not in epithelium , while stroma and epithelium displayed an intense immunostaining in cg and ga for both proteins . conclusion . these results have provided evidence that galectin-1 and mainly annexin - a1 are overexpressed in both gastritis and gastric cancer , suggesting a strong association of these proteins with chronic gastric inflammation and carcinogenesis .	chronic inflammation has been recognized as a process that can trigger cancer , due to the host immune response with local expression of cytokines , chemokines , adhesion molecules , and pro- and anti - inflammatory proteins that stimulate processes such as proliferation , survival , cell migration , and neovascularization . the strongest association between chronic inflammation and malignancy is observed in gastric cancer induced by helicobacter pylori infection . the inflammatory process resulting from h. pylori infection triggers a cascade of events initialized by chronic gastritis that evolves to gastric atrophy , intestinal metaplasia , dysplasia , and finally carcinoma . this bacterium is present in 90% of all chronic gastritis patients and in 77% of noncardia gastric cancers . both the intrinsic factors of the host and the bacterial virulence are associated with the development of gastric cancer induced by h. pylori . among the bacterial genes , there is caga , which is detected in about 63% of patients with gastric cancer . the caga protein is internalized by the host epithelial cells , disrupting the cell cycle and inducing cell invasion through the activation of matrix metalloproteases . bacterial lipopolysaccharides activate several cell processes , including expression of annexin - a1 ( anxa1 ) and galectin-1 ( gal-1 ) , which are both anti - inflammatory proteins . during the inflammatory response , anxa1 is translocated from the cell cytoplasm to the membrane , resulting in a decrease in the transmigration of inflammatory cells to the site of injury . furthermore , anxa1 plays its anti - inflammatory role by inhibiting the activities of phospholipase a2 and inducible nitric oxide synthase . it is also associated with modifications of the cytoskeleton , transport of molecules , ion flux , differentiation and migration , cell growth , and apoptosis . galectin-1 ( gal-1 ) , a member of a family of carbohydrate - binding proteins , may act in the same manner as anxa1 in the transmigration of inflammatory cells , being important also in t - cell apoptosis by binding to t - cell receptors , thus triggering the faz / caspase cascade . it also contributes to different events associated with carcinogenesis , including tumor transformation , cell cycle regulation , apoptosis , cell adhesion , migration , and inflammation [ 13 , 14 ] . nevertheless , there are only a few studies in the literature that evaluated the expression pattern of those anti - inflammatory mediators in gastric mucosa , but their specific functions are unclear . while in gastric cell lines the gal-1 protein expression increased [ 15 , 16 ] , in gastric adenocarcinoma was observed low expression in tumor cells . in turn , anxa1 was studied in gastric tissue with discrepant findings , such as increased expression during gastric mucosal damage healing , loss of expression in metastatic gastric cancers , higher expression in diffuse - type gastric cancer compared to the intestinal type , and decreased expression in gastric adenocarcinoma , but with positive staining in advanced stage and peritoneal dissemination . thus , further research in this field might improve our understanding of the possible role of those anti - inflammatory proteins in carcinogenesis , which is at present fairly limited and controversial . moreover , such studies may lead to the possible identification of anxa1 and gal-1 as potential biomarkers in gastric cancer progression . therefore , the aim of this study was to investigate the relative expression levels of anxa1 and lgals1 mrna by quantitative real - time pcr ( qpcr ) and the expression of both proteins by immunohistochemical assay in inflammatory gastric lesions such as chronic gastritis compared to gastric cancer . we also investigated a possible relationship between mrna expression levels and h. pylori infection and its caga virulence genotype in both lesions evaluated . gastric biopsies were obtained from seventy ( 70 ) individuals submitted to endoscopy at the endoscopy service of the hospital de base and at rio preto endoscopy center , both in so jos do rio preto , sp , brazil . the histopathological data were supplied , respectively , by the legal medicine department and by the pathology service and the institute of anatomical pathology and cytopathology ( iapc ) , in the same city . for each individual , three ( 03 ) biopsy samples from the antrum region were collected for molecular studies and one for immunohistochemical analysis . the gastric adenocarcinoma ( ga ) group comprised 20 individuals ( 14 male and 6 female ; mean age 63.4 14 years ) with a histopathologically confirmed diagnosis of gastric adenocarcinoma . among the studied samples , 12 cases were diagnosed as intestinal - type adenocarcinoma ( igc ) and 8 as diffuse - type adenocarcinoma ( dgc ) . the chronic gastritis ( cg ) group was composed of 40 individuals ( 18 male and 22 female ; mean age 52.5 15 years ) with a histopathologically confirmed diagnosis of chronic gastritis . the biopsies for the control ( c ) group were obtained from 10 healthy individuals ( 7 male and 3 female ; mean age 35 10.8 years ) with no dyspeptic gastric complaints and diagnosed as histopathologically normal gastric mucosa . epidemiological data on the study population were collected using a standard interviewer - administered questionnaire , with questions about current and past occupation , smoking habits , alcohol intake , and family history of cancer . none of the 70 subjects were under antibiotic or anti - inflammatory treatment neither radiotherapy nor chemotherapy . about 60% of all patients in the ga group were smokers and 40% were drinkers , while all patients in the cg and c groups were nonsmokers and nondrinkers . smokers were defined as individuals who consumed at least 100 cigarettes during their lifetime , and alcohol consumers were those who drank more than four times a week . the research ethics committee of the participating institution approved this research ( cep ibilce / unesp number 058/09 ) , and written informed consent was obtained from all individuals studied . soon after collection , the biopsies were stored in rna later solution ( applied biosystems ) at 20c , to preserve their integrity until rna extraction . the nucleic acid extraction was performed according to the protocol accompanying the reagent trizol ( invitrogen ) that allows the simultaneous extraction of rna and dna . rna and dna concentrations were determined in a nanodrop nd1000 spectrophotometer ( uniscience ) by measuring absorbance at 260 and 280 nm . dna samples were stored at 20c and used for the molecular diagnosis of h. pylori . afterwards , reverse - transcription ( rt ) pcr was performed in an automated thermocycler , using 2.5 g of total rna in the presence of 1.25 l of oligo - d(t)16 ( 0.5 g/l ) , 2.0 l of rnase inhibitor ( 80 u/l ) , and a high capacity cdna archive kit ( applied biosystems ) , in a total volume of 50 l , according to the manufacturer 's instructions . the reactions were carried out for 10 minutes at 25c , followed by 120 minutes at 37c . the integrity of all cdna preparations was tested by a pcr assay of a 613 bp actb ( -actin ) gene fragment , used as control for abundant transcripts , whose primer sequences were f : 5-ggcatcgtgatggactcc-3 and r : 3-gctggaaggtggacagcg-5. to determine the presence of h. pylori infection , dna samples were subjected to a multiplex pcr reaction containing primers for the bacterial gene hpx and for cyp1a1 ( human housekeeping gene , which attests the integrity of the dna ) . in summary , we used 5.0 l of 10x buffer , 5.0 l of dntps ( 1.23 mmol / l , invitrogen ) , 2.0 l mgcl2 ( 25 mmol / l ) , 2.0 l of primers ( 10 nmol/l , invitrogen ) , 24.5 l of dh2o , 5.0 l of genomic dna , and 0.5 l of taq dna polymerase ( 5 u/l , invitrogen ) . the material was processed in an automated thermocycler and was initially subjected to a temperature of 94c for 5 minutes for denaturation . subsequently , it was subjected to 40 amplification cycles at 94c for 45 seconds , at 60c for 30 seconds , and at 72c for 90 seconds , followed by a final extension cycle of 7 minutes at 72c . fragments of 150 bp and 226 bp corresponding to genes cyp1a1 and hpx , respectively , were observed . the h. pylori - positive samples were then subjected to a second pcr run , to investigate the virulence genotype caga of the bacterium . the parameters used were the same as for the previous reaction , except the annealing temperature , which in this case was 52c . the product was visualized on 1.0% agarose gel stained with ethidium bromide , and a 232 bp fragment was observed . the relative quantification q - pcr assay for anxa1 and lgals1 mrna expression was performed in an abi prism 7300 sequence detector system ( applied biosystems , foster city , ca , usa ) , according to the instructions for the sybr green pcr core reagent ( applied biosystems ) , using primers specific for genes anxa1 and lgals1 . gene actb was used as endogenous control ( reference gene ) of the reaction , because it had shown the lowest variation compared to -tubulin and 2-microglobulin genes in a previous study . the expression levels of the anxa1 , lgals1 , and actb mrna were tested in triplicate ( cdna from the same rt reaction , but in separated wells ) . samples of normal gastric mucosa were mixed to form a pool that was used as a calibrator ( standard sample ) . the q - pcr assays were performed in a total volume of 50 l , containing 10 l of sybr green master mix ( applied biosystems ) , 25 ng of cdna , and 0.4 m of anxa1 and 0.5 m lgals1 primers . after initial incubation at 50c for 2 min to allow uracil - n - glycosylase ( ung ) digestion and at 95c for 10 min to activate the amplitaq gold dna polymerase ( both provided by the universal pcr master mix ) , the samples were amplified by subjecting them to 40 biphasic cycles of 95c for 15 sec and 60c for 1 min . the fluorescence signal was measured in the extension phase of the pcr reaction , and a threshold value ( ct ) of fluorescence in the exponential part of the amplification curve was selected . the larger the quantities of the material at start , the lower the ct values . relative quantification ( rq ) of genes anxa1 and lgals1 was obtained as described by pfaffl and normalized with the -actin control reference gene and normal gastric mucosa . deparaffinized sections ( 4 m ) were incubated in citrate buffer , ph 6.0 , at 96c for 30 minutes , washed with distilled water , incubated with 3% hydrogen peroxide in methanol ( 30 minutes ) , and washed in phosphate - buffered saline ( pbs , ph 7.4 ) . the primary antibodies rabbit polyclonal anti - gal-1 and rabbit polyclonal anti - anxa1 ( zymed laboratories , cambridge , uk ) were diluted to 1 : 500 or 1 : 2000 , respectively , in 1% bovine serum albumin ( bsa ) and applied overnight at 4c . as negative controls , fragments were then washed in pbs , incubated with the universal lsab kit / hrp secondary antibody ( dako , usa ) according to the manufacturer 's protocol , washed in pbs , and developed with 3,3-diaminobenzidine in chromogen solution ( dako , usa ) . the sections were washed thoroughly in distilled water , counterstained with hematoxylin and mounted on glass slides . densitometric analysis for anxa1 and gal-1 immunostaining was performed using an arbitrary scale from 0 to 255 with the axiovision software on a zeiss - axioskop ii light microscope , and the data were expressed as mean se . fisher 's exact test was used to determine if there were significant differences between groups regarding the presence of bacteria and the caga genotype , the histological type of tumor , and the gender . the data obtained from mrna quantification were expressed as mean sd . to assess differences in the mrna relative expression levels between the groups , we used the nonparametric mann - whitney test . to evaluate the association between gene expression and the presence of the bacterium and caga genotype , histological type of tumor , gender , smoking , and drinking the mean densitometry analysis results obtained for proteins anxa1 and gal-1 were compared by anova , followed if significant by the bonferroni test . the frequencies of cases with positive molecular diagnosis for h. pylori and genotype caga in groups cg and ga are presented in table 2 . all 10 samples of normal mucosa were confirmed by molecular testing as h. pylori negative . out of a total of 60 samples from the case groups , 45% were h. pylori positive : 40% ( 16/40 ) in the cg group and 55% ( 11/20 ) in the ga group , with no significant difference between the groups ( p = 0.29 ) . regarding the genotype caga , 48% of h. pylori - positive samples were caga positive : 62.5% ( 10/16 ) in the cg and 27.3% ( 3/11 ) in the ga group . again , there was no significant difference between the groups ( p = 0.12 ) . the relative expression levels of anxa1 mrna , after normalization with the actb reference gene and comparison with the normal mucosa , were increased in 90% ( 36/40 ) of the cg cases ( mean rq = 4.26 2.03 ) and in 80% ( 16/20 ) of the ga cases ( mean rq = 4.38 4.77 ) , so there was no statistically significant difference between the groups ( p = 0.33 ) . for lgals1 , the mrna relative expression values found were lower ; only the ga group showed overexpression in 60% ( 12/20 ) of the cases the cg group showed constitutive expression ( mean rq = 0.43 3.13 ) , with only 7.5% ( 3/40 ) of the cases presenting increased expression . thus , the mean level of lgals1 mrna expression was significantly higher in the ga than in the cg group ( p < 0.01 ) ( table 3 and figure 1 ) . in another analysis , we investigated the ga group for a possible association between anxa1 and lgals1 mrna expression and risk factors such drinking , smoking , and histological type of gastric cancer ( table 4 ) , but no association was observed . in addition , comparing the variables , gender , h. pylori infection , and caga+ genotype , in the cg and the ga groups ( table 5 ) , a significant difference was found in the ga group for the gender and the mean level of anxa1 expression ( p = 0.04 ) , due to a 2 times greater expression of this gene in the females than in the males of this group . likewise , the caga+ genotype also showed an association with the anxa1 expression level in the ga group , due to a higher mrna expression in the caga - positive compared to the caga - negative cases ( mean rq 6.40 versus 2.77 , p < 0.01 ) . none of the other investigated factors appears to be associated with the levels of anxa1 and lgals1 mrna . protein expression was evaluated in normal mucosa , cg , intestinal ( igc ) , and diffuse- ( dgc- ) type gastric cancer . modest expression of anxa1 and gal-1 was observed in the stroma of normal mucosa , while the epithelium did not show any expression of these proteins ( figures 2(a ) and 3(a ) , resp . ) . however , in the inflammatory process of cg mucosa , intense immunostaining of anxa1 and gal-1 was seen in the basal portion of the epithelium ( figures 2(b ) and 3(b ) , resp . ) . positive immunostaining for anxa1 and gal-1 was also observed in dgc ( figures 2(c ) and 3(c ) , resp . ) and igc ( figures 2(d ) and 3(d ) , resp . ) tumor cells . in some areas of gastric cancer samples the mean optical densitometry values of anxa1 and gal-1 expression are presented in figures 2(e ) and 3(e ) , respectively . the anxa1 mean density was 90.79 for normal mucosa ( c group ) , while for cg and ga , it was , respectively , 168.57 and 190.20 . thus , a significant difference was found comparing the normal mucosa with the cg and ga groups ( p < 0.01 for both ) . the mean density of the gal-1 protein was lower in all three groups ( 86.52 , 136.97 , and 146.30 in c , cg , and ga , resp . ) , showing a statistically significant difference between the normal mucosa and the cg and ga groups ( p < 0.01 for both ) . in another densitometry analysis to determine the cytoplasmic and nuclear immunoreactivity of anxa1 separately in all groups , although observed nuclear immunostaining mainly in gastric cancer , the data obtained in our results did not show a statistically significant difference between normal mucosa and cg ( p = 0.19 ) and normal mucosa and ga ( p = 0.18 ) ( data not shown ) . in the present study , we evaluated anxa1 and gal-1 anti - inflammatory proteins and mrna expression in a group of precursor lesions such as chronic gastritis compared to gastric cancer and their association with risk factors . among the risk factors , we investigated the presence of h. pylori in both lesions and its virulence genotype caga , since this bacterium often triggers the progression of the gastric carcinogenesis cascade . to the best of our knowledge , this is the first study that has evaluated the expression of anxa1 and gal-1 in chronic gastritis . we have found a high relative expression of anxa1 mrna already in the cg inflammatory process , in which 90% of cases showed an increased expression level ( mean rq = 4.26 ) , which became 3 to 8 times higher after normalization with the actb reference gene and comparison with normal mucosa . similarly , in the ga group , 80% of cases presented upregulated expression levels , which were 3 to 9 times higher ( mean rq = 4.38 ) than that in normal mucosa . for lgals1 , our study showed a slightly increased relative expression only in 60% of the ga cases ( mean rq = 2.44 ) , with an increase of 2 to 7 times , but in chronic gastritis the mean value was low ( mean rq = 0.43 ) . in general , the immunohistochemical analysis confirmed the results of mrna expression by qpcr , although in cg the relative expression levels of lgals1 mrna was not equivalently increased as protein expression . studies on the expression of anxa1 mrna in neoplastic processes are still limited and the results are conflicting . for example , loss of expression was found in squamous cell carcinoma of the esophagus , prostatic adenocarcinoma , sinonasal adenocarcinoma , larynx , and breast cancer . on the other hand , overexpression has been reported in colorectal adenocarcinoma , urothelial carcinoma , lung adenocarcinoma , and oral cancers , thus suggesting that changes in the expression levels of anxa1 may be related to the tissue or tumor type . martin et al . reported that normal gastric mucosa shows weak expression of the protein anxa1 , while in the ulcer healing process the expression was increased , promoting the reduction of the ulcer . in contrast , yu et al . observed overexpression of both gene and protein in normal mucosa but loss of expression in 64% of primary gastric tumors , mainly correlated with advanced stage and metastasis . more recently , zhu et al . observed that anxa1 protein is expressed in both gastric adenocarcinoma ( 45% ) and normal tissues ( 69% ) , but with different subcellular distribution . similar results were reported by cheng et al . that observed high anxa expression , both mrna and protein , associated with metastasis , invasion , and poor survival in gastric cancer patients . the authors also proposed a new mechanism of how anxa1 regulates the gastric cancer cell invasion through activation fpr / erk / itgb1bp1 pathway . in the present study , the immunohistochemical analysis for anxa1 showed modest immunostaining in stromal cells of normal mucosa and intense expression in stroma and epithelium of both cg and ga , thus confirming the results of the mrna expression analysis . the assay used in this study did not allow a clear differentiation of the cellular localization of the protein , although positive immunostaining was observed in the cytoplasm of epithelial cells and the nucleus of cancer cells , with lower intensity and frequency in chronic gastritis . however , when the densitometry analysis to determine cytoplasmic and nuclear immunoreactivity of anxa1 separately was performed , the data obtained did not show a statistically significant difference among normal mucosa , chronic gastritis , and gastric adenocarcinoma . although , there are some reports that show translocation of anxa1 during carcinogenesis , as alves et al . that showed 87.5% positivity for anxa1 in larynx tumors and increased immunoreactivity in the membrane compared to the cytoplasm and the nucleus . however , compared to the normal tissue , the nuclear and cytoplasmic expression was lower . in esophageal carcinoma , liu et al . found anxa1 translocation from the cellular to the nuclear membrane . while in gastric adenocarcinoma anxa1 showed positive nuclear staining correlated with advanced disease stage and peritoneal dissemination but in normal tissues was predominantly localized in the cytoplasm . in addition , weak nuclear staining also occasionally occurred in sporadic cells of gastric tumors ( 14/118 cases ) evaluated by cheng et al . . but , in general , the anxa1 immunostaining was mainly cytoplasmatic in the epithelial cells . changes in the expression pattern of anxa1 must be related with factors that influence its translocation and export , such as the cellular concentration of calcium , considering that , when the intracellular calcium level is increased , anxa1 is translocated to membranes . furthermore , it is reported that protein phosphorylation is also required for this process . in tumor cells , calcium and other factors however , this relationship needs to be better understood and may be the connection between inflammation , signal transduction , differentiation , and cellular transport in cancer . to date , gal-1 is involved in various important aspects of carcinogenesis , so mrna and protein expressions have been examined in several types of cancer . overexpression has been reported in colorectal cancer , hodgkin 's lymphoma , squamous cell carcinomas of the larynx , and carcinomas of the hypopharynx . conversely , there is also a report of low expression , as in chronic inflammation of nasal polyposis . two research groups evaluated the expression of gal-1 in gastric cell lines and both agreed that this protein is overexpressed in tumor cells . chen et al . showed higher expression of the protein in tmc-1 compared to sc - m1 cells , proposing gal-1 as a biomarker for metastasis . investigated ags cells infected by h. pylori and observed high expression levels of gal-1 in this cell type . recently , estofolete et al . observed by immunostaining that both gal-1 and -3 were highly expressed in an experimental model of n - methyl - n-nitro - n - nitrosoguanidine- ( mnng- ) induced gastric carcinogenesis . the immunohistochemistry assay performed showed modest staining for gal-1 in the stroma of normal mucosa , while in cg and ga the staining was stronger in stroma and epithelium . in contrary , the lgals1 mrna levels were lower in the cg group in comparison with the ga group . several studies have shown lack of correlation between rna expression and protein expression profiles using different methodologies . some authors state that in fact the use of mrna expression patterns is insufficient for understanding the expression of protein products , as additional posttranscriptional mechanisms , including protein translation , posttranslational modification , and degradation , may influence the level of a protein present in a given cell or tissue [ 4750 ] . the comparison between the cg and ga groups regarding anxa1 and lgals1 mrna levels and risk factors did not show any association with the lgals1 mrna levels . however , a positive association was observed between overexpression of anxa1 and female gender in the ga group , since in women the mrna expression was twice as high as in men ( mean rq = 6.67 versus 3.25 ) , and the h. pylori - caga+ infection showed an mrna mean level approximately twice higher in patients infected by caga+ strains ( mean rq = 6.40 versus mean rq = 2.77 ) . despite the relevance of the study , it should be considered some limitations due to the reduced number of cases in the subgroup stratification , which should be interpreted with cautions . it is well known that there is sexual dimorphism in the immune and inflammatory responses in humans . women produce more vigorous cellular and humoral reactions , are more resistant to certain infections , and suffer a higher incidence of autoimmune diseases than males . it has also been suggested that the anxa1 expression may differ in several types of cancer due to hormonal influence . ang et al . conducted a very elegant study on the influence of anxa1 in mcf-7 breast cancer cells , in which they showed that 17 -estradiol ( active metabolite of estrogen ) regulates anxa1 expression . moreover , 17 -estradiol was shown to activate cyclic - amp- ( camp- ) responsive element ( cre ) binding ( creb ) proteins to induce a transcriptional activity . the promoter region of anxa1 was examined and found to contain a similar , near identical , 8-nucleotide sequence ( tgatgtca ) to the cre consensus sequence ( tgacgtca ) . , those authors proposed another theory to explain the connection between estrogen and anxa1 levels . elevated 17 -estradiol activates the erk1/2 pathway , increasing cell proliferation , and this serves as a sign to stimulate anxa1 transcription in order to reduce the proliferative state . then , anxa1 upregulates p21 , which has antiproliferative properties , and reduces the proliferation caused by the activation of erk1/2 by estrogen . however , in our study , in the ga group the mean age of women was elevated ( 67.4 18.4 years ) , which characterizes the postmenopausal phase that have decreased estrogen levels , thus not justifying the relation between increased expression of anxa1 and estrogen . the relationship between the presence of virulence factor caga and gastric carcinogenesis is well documented . western populations infected with caga - positive strains generally have an accentuated inflammatory response , with increased risk of developing peptic ulcer and stomach cancer . the phosphorylation of the caga protein activates shp-2 ( protein tyrosine phosphatase ) , which then inhibits the fak ( focal adhesion kinase ) , an enzyme that modulates adhesion , migration , and cell survival . consequently , the decline in the activity of fak triggers a rearrangement of the cytoskeleton known as the hummingbird phenotype , characterized by elongation and spreading of host cells . furthermore , caga participates in cell signaling by activating the pik3ca and kras pathways , erk ( mapk ) [ 55 , 56 ] , and mek / erk and jak1 signaling pathway in gastric cancer cells . to our knowledge , there are no reports about the relationship to the caga virulence factor and the expression of anxa1 and gal-1 anti - inflammatory proteins . however , lin et al . observed overexpression of anxa4 in tumor cells of patients infected with h. pylori and in gastric cancer scm-1 cells after h. pylori infection . recently , lin et al . observed that infection by h. pylori induced a change in anxa1 and anxa4 localization , causing a translocation from the cytoplasm to the plasma membrane , probably for epithelial cell membrane repair in the consequence of h. pylori - generated membrane disruptions . so , due to the action of caga bacterial protein in different cell signaling pathways , it is possible that it may also contribute to activation of anxa1 expression mainly , considering that this protein plays a key role as intracellular ca flux , modifications of the cytoskeleton , differentiation and migration , cell growth , and apoptosis . in conclusion , this study showed overexpression of both anxa1 mrna and protein already in a precursor lesion such as cg , similar to ga , in which higher expression levels were observed in h. pylori - caga+ cases , suggesting upregulation of this gene in early stages of gastric carcinogenesis . in turn , lgals1 ( mrna or protein ) was slightly overexpressed in both lesions , indicating also its participation in gastric carcinogenesis . however , as in several types of cancers the role of these proteins is not yet fully understood , further investigations are needed to help clarify the molecular mechanisms by which they act in this kind of lesion .
objectives : there is a direct relationship between the regression of left ventricular hypertrophy ( lvh ) and a decreased risk of mortality . this investigation aimed to describe the effects of anti - hypertensive drugs on cardiac hypertrophy through a meta - analysis of the literature.methods:the medline ( via pubmed ) , lilacs and scielo databases were searched using the subject keywords cardiac hypertrophy , antihypertensive and mortality . we aimed to analyze the effect of anti - hypertensive drugs on ventricle hypertrophy.results:the main drugs we described were enalapril , verapamil , nifedipine , indapamina , losartan , angiotensin - converting enzyme inhibitors and atenolol . these drugs are usually used in follow up programs , however , the studies we investigated used different protocols . enalapril ( angiotensin - converting enzyme inhibitor ) and verapamil ( ca++ channel blocker ) caused hypertrophy to regress in lvh rats . the effects of enalapril and nifedipine ( ca++ channel blocker ) were similar . indapamina ( diuretic ) had a stronger effect than enalapril , and losartan ( angiotensin ii receptor type 1 ( at1 ) receptor antagonist ) produced better results than atenolol ( selective 1 receptor antagonist ) with respect to lvh regression.conclusion:the anti - hypertensive drugs induced various degrees of hypertrophic regression .	this phenomenon is a result of protein synthesis and either the incorporation of new units into contractile myocytes or the addition of new sarcomeres.3 nevertheless , the benefits of this process are limited by the rate of maintenance , which promotes deleterious changes characterizing this feature as uniquely unfavorable.4 the mechanisms leading to myocardial injuries59 and hypertrophy1012 during physiological or pathological states are different . for instance , the pathological hypertrophy results from the interaction of mechanical forces and neural and hormonal factors . hemodynamic overload promotes myocyte stretch , the release of intracellular calcium , the activation of calcineurin and the induction of gene expression reprogramming.1 in athletes , left ventricular hypertrophy ( lvh ) is regarded as a physiological response that results from a hemodynamic overload ; this physiological response does not result in the harmful effects of hypertension development or other heart diseases.1012 the literature explains physiological lvh as the result of a volume and pressure overload that results from intense physical training ( the hemodynamic stimulus ) , which is not necessarily accompanied by neural and humoral changes.13 despite some initial benefits of physiological hypertrophy , it is difficult to determine the time at which physiological hypertrophy becomes pathological1 and represents a significant morbidity and mortality risk . in other words , it is difficult to pinpoint exactly when , during the natural development of hypertension , cardiac hypertrophy raises an individual s risk of experiencing a cardiovascular event by a reported factor of six to eight times.3 there are well - known associations between lvh and several conditions , such as obesity , diabetes , myocardial infarction and even mild elevation in blood pressure,1 as well as other cardiovascular risk factors that may be associated with increased cardiac mass regardless of ventricular pressure . therefore , lvh is a powerful predictor of cardiovascular morbidity and mortality.3 clinical and experimental studies have reported that several pharmacological , hemodynamic and non - hemodynamic factors are able to induce , reverse and prevent lvh,1416 interstitial fibrosis17 and the progression of atherosclerotic effects on myocytes.18 clinically , cardiac hypertrophy is associated with increased adverse events , including stroke , chronic renal failure , ventricular dysfunction , ventricular arrhythmias and sudden death.3 cardiac hypertrophy is also associated with a higher prevalence of coronary disease and is related to endothelial dysfunction . this dysfunction may lead to myocardial ischemia , even in the absence of epicardial artery obstruction , by reducing coronary flow reserves due to various hemodynamic factors , such as a minimal reduction in coronary resistance , systolic coronary perivascular compression , collagen deposition , fibrosis and a lower production of local vasodilator substances , such as nitric oxide.19 after adjusting for other risk factors , koren and coworkers20 reported a positive relationship between ventricular mass and geometry with the morbidity and mortality of patients who did not have complicated hypertension.20 the framingham heart study , published by levy et al21 included 3,220 healthy subjects aged 40 years or older . this study demonstrated that estimating left ventricular mass by echocardiography can provide prognostic information about cardiovascular risk and can predict negative cardiovascular - related clinical events , including death.21 moreover , lvh increased the risk of major cardiovascular events in 40% of the patients enrolled in the study.21 from these previous investigations , we infer that lvh constitutes an ominous risk factor for cardiovascular disease and that the magnitude of this risk is directly related to morbidity and mortality rates . therefore , in this study , we endeavored to describe the effects of anti - hypertensive drugs on cardiac hypertrophy by a meta - analysis of previous studies . the medline ( via pubmed ) , lilacs and scielo databases were searched using the following subject keywords : cardiac hypertrophy , antihypertensive and mortality . function on pubmed , lilacs and scielo , which allowed us to search the references of the studies that were retrieved during our search . publications were included in our review if either their titles or abstracts were available in english or portuguese and suggested any effect ( i.e. , beneficial or malefic ) or lack thereof from an anti - hypertensive treatment on cardiac hypertrophy . publications were excluded if the treatment was limited to a particular technique or if the population received only one specific procedure ( drug treatment ) associated with a disease state or an age group . other studies on anti - hypertensive treatment for hypertensive patients that offered additional relevant information found in the same database were also examined . each publication was reviewed to identify the author(s ) , study period and data source , which influenced us to include the reference in the study . we excluded investigations in which treatment was limited to a particular method and those studies in which the study population was limited to patients who were undergoing a particular procedure or an associated disease state . in table 1 , we summarize studies that evaluated the relationship between anti - hypertensive drugs and cardiac hypertrophy . the investigations included in table 1 demonstrate the relevance of angiotensin - converting enzyme ( ace ) inhibitors14,15,2224 and angiotensin antagonist25 treatment for cardiac hypertrophy regression . the high prevalence of lvh and the high rate of complications of its condition necessitate a thorough understanding of the mechanisms involved in its etiology and development , as well as the importance of prevention and regression , to optimize and prevent the deleterious consequences . thus , it is important to clarify the following issues , which are described below . several published reports have provided data indicating that anti - hypertensive drugs promote ventricular hypertrophy regression and myocardial repair . in particular , ace inhibitors have shown promising cardio - protective effects in experimental models of hypertensive heart disease;22 specifically , these inhibitors improve micro - vascular ischemia , ventricular function and arrhythmias.1416 , 2329 we have previously reported that enalapril14 ( ace inhibitor ) and verapamil23 ( ca channel blocker ) provide the same cardio - protector response in an isoproterenol - induced lvh model in rats.28 to evaluate the effects of lvh and its regression , a previous study evaluated 151 patients with hypertension and lvh progression detected by echocardiography . the patients were followed for an average of 10 years+1.4 years , and the researchers noted a decrease in the non - fatal events rate following reduction of lvh , even after correcting for other risk factors . conversely , the occurrence of these events increased when cardiac hypertrophy increased or remained stable.23 another clinical investigation examined 430 patients with essential hypertension ( also called primary or idiopathic hypertension ) and related changes in the left ventricular mass with the occurrence of cardiovascular events . treatments consisted of medication and lifestyle changes . at the 2 years and 9 months follow - up assessment , the prevalence of lvh by echocardiography was 26% per year , and the rate of cardiovascular events was 3.9% per year . for individuals who showed an improvement in lvh , in the groups that experienced no change or a worsening in lvh , the rate of cardiovascular events was 6.27 events per 100 patients per year . these researchers also noted that this association did not depend on the initial ventricular mass , clinical parameters or monitored baseline blood pressure . their final conclusion was that for primary hypertension , the decrease in ventricular mass during treatment is a favorable prognostic marker for subsequent morbid events . with the exception of minoxidil and hydralazine , which are peripheral vasodilators , the other anti - hypertensive drugs provided full or partial lvh regression.28 not all classification , prospective and randomized studies that have evaluated the effectiveness of various drugs with respect to left ventricular mass reduction have reported evidence of a difference between their effects on lvh regression.23 however , in view of the different effects of the drugs , those drugs that depend on neural and humoral mechanisms , especially as reported in monotherapy studies , provide evidence that there may be several ways in which the drugs properties could reduce ventricular mass . in the literature , there are few monotherapy treatment meta - analyses . in addition , different groups report contradictory results.23 most studies have used anti - hypertensive drugs ( i.e. , beta - blockers and diuretics ) and reported 58% reductions of the left ventricular mass , while the use of ace inhibitors and angiotensin at1 blockers resulted in a 13% reduction . the difference between different anti - hypertensive drugs regarding their ability to reverse lvh may be determined by neural and humoral factors.30 because lvh regression is an important intermediate objective of anti - hypertensive therapy , several trials and meta - analyses have compared the effects of these drugs on ventricular hypertrophy , but the usefulness of these studies is limited due to inadequate designs and methodological problems.32 the preserve ( prospective randomized enalapril study evaluating regression of ventricular enlargement),33 live ( indapamide sustained release versus enalapril)34 and life ( losartan intervention for endpoint in hypertension)25 studies represent a new generation of well - planned trials comparing different anti - hypertensive drugs . these investigations revealed that during regression of lvh , the effects of nifedipine and enalaprilat are similar ( preserve ) ; indapamine has a stronger effect than does enalapril ( live ) ; and losartan treatment has a stronger effect than atenolol ( life ) . the findings of these three studies conflict with the findings of some previous meta - analyses , and as such , these researchers have suggested that the effects of anti - hypertensive drug must be evaluated individually on their important intermediate targets , such as lvh , with appropriate patient populations . the extrapolation of the lvh regression effects based on the anti - hypertensive drug classification may lead to clinical mistakes and needs to be carefully reconsidered.23 a decrease in blood pressure may reduce a ventricular mass . in addition to clinical studies , experimental studies have demonstrated that lvh prevention and/or regression is independent of blood pressure . for instance , our group investigated isoproterenol - induced lvh in albino rats , which typically leads to hypertrophy without an increase in the after - load , and reported lvh regression by gavage administration of enalapril15 , 16 , 28 , 31 and verapamil.23 , 30 moreover , studies in spontaneously hypertensive rats , a rat strain commonly used in our laboratory3539 , demonstrated that although various anti - hypertensive drugs may control systemic hypertension , lvh may be influenced by many , but not all , anti - hypertensive agents . alpha - methyldopa , captopril , beta - blockers and calcium channel blockers promote the regression of hypertrophy , while other drugs , such as hydralazine and minoxidil , reduce blood pressure without influencing ventricular hypertrophy.23 however , the development of lvh in arterial hypertension may be explained primarily by an increased pressure overload and ventricular wall tension . the ratio of ventricular mass to pressure is often weak , even in relation to blood pressure monitored every 24 hours.3 notably , lvh may occur independently from changes in arterial pressure and be present even when hypertension does not develop.1 we may include the presence of others factors involved in this condition , such as lifestyle , anthropometric and demographic characteristics , genetic influences and neural and humoral factors.24 furthermore , in vivo , in vitro and genetic studies indicate that lvh development and its regression depends not only on the hemodynamic overload but also on other genetic , neural and humoral factors.9 humoral agents that may affect mitogenesis and non - myocyte cardiomyocytes have been identified ; these agents include the renin - angiotensin system , local norepinephrine , endothelin , transforming growth factor , insulin - like growth factor , bradykinin , prostaglandins and nitric oxide.23 the direct relationship between high blood pressure and lvh development has been questioned due to the fact that lvh development and its regression depends not only on the hemodynamic overload , forcing a review of the mechanisms involved in lvh development , such as the role of the sympathetic nervous system , the renin - angiotensin system , genetic factors , endothelin and endothelium.1 it is important to remember that the pathogenesis of lvh includes angiotensin ii receptors and membrane - stretched cardiomyocytes.2 several publications have reported that although anti - hypertensive drugs show non - uniform effects on lvh , there are no concomitant proportional decreases in blood pressure.13 , 20 conversely , anti - hypertensive drugs have promoted different effects on lvh , despite inducing similar reductions in blood pressure.4 those assumptions lead us to consider other mechanisms involved in lvh regression that are independent of blood pressure reduction . a decrease in cardiac mass may not depend only on daily arterial pressure measurement.23 recently , mayos et al.40 localized chromosome regions that harbor genetic variants that affect the diversity of electrocardiographic and echocardiographic lvh . they evaluated the genetic association of the sokolow - lyon voltage index and the cornell product index , the septal thickness and the ventricular wall , ventricular dimensions and left ventricular mass in 868 members of 224 british households , all items were evaluated n pairs and together . chromosomes 10 , 12 and 17 were the genetic loci involved and had the most important influence on lvh , as detected by the electrocardiogram.23 genetic factors may explain a substantial portion of the quantitative variability in the electrocardiographic and echocardiographic examinations due to hemodynamic and/or hormonal factors . however , the quantitative differences in lvh , known as phenotypes , may also be the result of individual differences besides those produced by the various anti - hypertensive drugs ( i.e. , beyond blood pressure ) . the high prevalence of lvh and the high rate of complications of its condition necessitate a thorough understanding of the mechanisms involved in its etiology and development , as well as the importance of prevention and regression , to optimize and prevent the deleterious consequences . thus , it is important to clarify the following issues , which are described below . several published reports have provided data indicating that anti - hypertensive drugs promote ventricular hypertrophy regression and myocardial repair . in particular , ace inhibitors have shown promising cardio - protective effects in experimental models of hypertensive heart disease;22 specifically , these inhibitors improve micro - vascular ischemia , ventricular function and arrhythmias.1416 , 2329 we have previously reported that enalapril14 ( ace inhibitor ) and verapamil23 ( ca channel blocker ) provide the same cardio - protector response in an isoproterenol - induced lvh model in rats.28 to evaluate the effects of lvh and its regression , a previous study evaluated 151 patients with hypertension and lvh progression detected by echocardiography . the patients were followed for an average of 10 years+1.4 years , and the researchers noted a decrease in the non - fatal events rate following reduction of lvh , even after correcting for other risk factors . conversely , the occurrence of these events increased when cardiac hypertrophy increased or remained stable.23 another clinical investigation examined 430 patients with essential hypertension ( also called primary or idiopathic hypertension ) and related changes in the left ventricular mass with the occurrence of cardiovascular events . treatments consisted of medication and lifestyle changes . at the 2 years and 9 months follow - up assessment , the prevalence of lvh by echocardiography was 26% per year , and the rate of cardiovascular events was 3.9% per year . for individuals who showed an improvement in lvh , the rate of cardiovascular events was 1.58 events per 100 patients per year . in the groups that experienced no change or a worsening in lvh , the rate of cardiovascular events was 6.27 events per 100 patients per year . these researchers also noted that this association did not depend on the initial ventricular mass , clinical parameters or monitored baseline blood pressure . their final conclusion was that for primary hypertension , the decrease in ventricular mass during treatment is a favorable prognostic marker for subsequent morbid events . with the exception of minoxidil and hydralazine , which are peripheral vasodilators , the other anti - hypertensive drugs provided full or partial lvh regression.28 not all classification , prospective and randomized studies that have evaluated the effectiveness of various drugs with respect to left ventricular mass reduction have reported evidence of a difference between their effects on lvh regression.23 however , in view of the different effects of the drugs , those drugs that depend on neural and humoral mechanisms , especially as reported in monotherapy studies , provide evidence that there may be several ways in which the drugs properties could reduce ventricular mass . in the literature , there are few monotherapy treatment meta - analyses . in addition , different groups report contradictory results.23 most studies have used anti - hypertensive drugs ( i.e. , beta - blockers and diuretics ) and reported 58% reductions of the left ventricular mass , while the use of ace inhibitors and angiotensin at1 blockers resulted in a 13% reduction . the difference between different anti - hypertensive drugs regarding their ability to reverse lvh may be determined by neural and humoral factors.30 because lvh regression is an important intermediate objective of anti - hypertensive therapy , several trials and meta - analyses have compared the effects of these drugs on ventricular hypertrophy , but the usefulness of these studies is limited due to inadequate designs and methodological problems.32 the preserve ( prospective randomized enalapril study evaluating regression of ventricular enlargement),33 live ( indapamide sustained release versus enalapril)34 and life ( losartan intervention for endpoint in hypertension)25 studies represent a new generation of well - planned trials comparing different anti - hypertensive drugs . these investigations revealed that during regression of lvh , the effects of nifedipine and enalaprilat are similar ( preserve ) ; indapamine has a stronger effect than does enalapril ( live ) ; and losartan treatment has a stronger effect than atenolol ( life ) . the findings of these three studies conflict with the findings of some previous meta - analyses , and as such , these researchers have suggested that the effects of anti - hypertensive drug must be evaluated individually on their important intermediate targets , such as lvh , with appropriate patient populations . the extrapolation of the lvh regression effects based on the anti - hypertensive drug classification may lead to clinical mistakes and needs to be carefully reconsidered.23 in addition to clinical studies , experimental studies have demonstrated that lvh prevention and/or regression is independent of blood pressure . for instance , our group investigated isoproterenol - induced lvh in albino rats , which typically leads to hypertrophy without an increase in the after - load , and reported lvh regression by gavage administration of enalapril15 , 16 , 28 , 31 and verapamil.23 , 30 moreover , studies in spontaneously hypertensive rats , a rat strain commonly used in our laboratory3539 , demonstrated that although various anti - hypertensive drugs may control systemic hypertension , lvh may be influenced by many , but not all , anti - hypertensive agents . alpha - methyldopa , captopril , beta - blockers and calcium channel blockers promote the regression of hypertrophy , while other drugs , such as hydralazine and minoxidil , reduce blood pressure without influencing ventricular hypertrophy.23 however , the development of lvh in arterial hypertension may be explained primarily by an increased pressure overload and ventricular wall tension . the ratio of ventricular mass to pressure is often weak , even in relation to blood pressure monitored every 24 hours.3 notably , lvh may occur independently from changes in arterial pressure and be present even when hypertension does not develop.1 we may include the presence of others factors involved in this condition , such as lifestyle , anthropometric and demographic characteristics , genetic influences and neural and humoral factors.24 furthermore , in vivo , in vitro and genetic studies indicate that lvh development and its regression depends not only on the hemodynamic overload but also on other genetic , neural and humoral factors.9 humoral agents that may affect mitogenesis and non - myocyte cardiomyocytes have been identified ; these agents include the renin - angiotensin system , local norepinephrine , endothelin , transforming growth factor , insulin - like growth factor , bradykinin , prostaglandins and nitric oxide.23 the direct relationship between high blood pressure and lvh development has been questioned due to the fact that lvh development and its regression depends not only on the hemodynamic overload , forcing a review of the mechanisms involved in lvh development , such as the role of the sympathetic nervous system , the renin - angiotensin system , genetic factors , endothelin and endothelium.1 it is important to remember that the pathogenesis of lvh includes angiotensin ii receptors and membrane - stretched cardiomyocytes.2 several publications have reported that although anti - hypertensive drugs show non - uniform effects on lvh , there are no concomitant proportional decreases in blood pressure.13 , 20 conversely , anti - hypertensive drugs have promoted different effects on lvh , despite inducing similar reductions in blood pressure.4 those assumptions lead us to consider other mechanisms involved in lvh regression that are independent of blood pressure reduction . a decrease in cardiac mass may not depend only on daily arterial pressure measurement.23 recently , mayos et al.40 localized chromosome regions that harbor genetic variants that affect the diversity of electrocardiographic and echocardiographic lvh . they evaluated the genetic association of the sokolow - lyon voltage index and the cornell product index , the septal thickness and the ventricular wall , ventricular dimensions and left ventricular mass in 868 members of 224 british households , all items were evaluated n pairs and together . chromosomes 10 , 12 and 17 were the genetic loci involved and had the most important influence on lvh , as detected by the electrocardiogram.23 genetic factors may explain a substantial portion of the quantitative variability in the electrocardiographic and echocardiographic examinations due to hemodynamic and/or hormonal factors . however , the quantitative differences in lvh , known as phenotypes , may also be the result of individual differences besides those produced by the various anti - hypertensive drugs ( i.e. , beyond blood pressure ) . current medical literature shows the direct relationship between lvh and mortality , as well as the relationship between lvh regression and decreasing mortality . the literature also provides evidence for hypertrophic variations in response to the effect of different anti - hypertensive drugs and differences in the hemodynamic patterns .
littoral cell angioma ( lca ) is a rare primary splenic tumor that is difficult to differentiate preoperatively from other benign and malignant splenic lesions . most of the cases present as multiple nodules in the spleen . we report a case of large solitary lca of the spleen , an uncommon presentation . lca should be considered in the differential diagnosis of multiple and solitary splenic lesions .	littoral cell angioma ( lca ) is a recently described rare primary vascular neoplasm , unique to the spleen . this neoplasm has characteristic morphological and immunophenotypic features that distinguish it from other vascular splenic tumors . most of the cases described in the literature were lcas composed of multiple nodules of variable sizes . we report a large solitary angioma in the spleen , an uncommon presentation of this rare neoplasm . a 58-year - old female patient presented to her primary care physician with chronic non - specific bilateral lower rib pain . routine laboratory tests were normal including hemoglobin , hematocrit , white blood cell , and platelet count . a contrast - enhanced computed tomogram ( ct ) scan was performed during the portal venous phase , the retrocardiac opacity seen on the chest radiograph turned out to be hiatal hernia . the spleen was enlarged with a large 6.6 cm 6.0 cm solitary mass [ figure 1 ] . there was diffuse , homogeneous enhancement on the lateral aspect of the mass and the medial aspect was hypoenhanced , probably due to a necrotic portion or a large nonenhanced vascular channel . to further characterize the mass , multiphasic magnetic resonance imaging ( mri ) the mri [ figure 2 ] showed the mass to be both t1 and t2 isointense with mild peripheral nodular and internal septal enhancement during the arterial phase with complete , diffuse , and mild enhancement of the mass during the portal venous phase , which remained enhanced until delayed images . the differential diagnoses considered were primary vascular neoplasms including atypical hemangioma ( as mass was not t2 hyperintense ) , hamartoma , and potential angiosarcoma . histological examination of the fragments of mass revealed anastomosing and congested sinusoidal channels with fibrous interstitial tissue and absence of normal red and white pulp . the lining cells were immunoreactive for cd 31 , factor viii , and cd 68 , and were negative with stains for cd8 and cd34 ( markers that are normally present on red pulp sinusoidal endothelium ) , consistent with diagnosis of lca . contrast - enhanced axial computed tomogram ( ct ) image obtained during portal venous phase shows a large mass ( arrows ) in the spleen , the mass is showing diffuse homogeneous enhancement on the lateral aspect and the medial aspect is hypoenhanced . a cyst in the liver is also demonstrated ( arrowhead ) . axial magnetic resonance imaging ( mri ) images at the level of mid spleen show a large mass ( arrows ) which is isointense to the rest of splenic parenchyma before contrast injection on ( a ) t1-weighted and ( b ) t2-weighted images . ( c ) it shows peripheral nodular and internal septal enhancement during arterial phase on t1-weighted fat suppressed pulse sequence . ( d ) diffuse enhancement during portal venous phase and ( e ) remains enhanced on delayed 3-min image . as 30% of lca cases have been reported to be associated with other malignancies , she underwent further investigations including ct chest and colonoscopy , which were normal . the differential diagnosis of splenic vascular tumor is broad and may represent benign ( hemangioma , hamartoma , lymphangioma ) , indeterminate ( lca , hemangioendothelioma , hemangiopericytoma ) , or malignant neoplasms ( angiosarcoma ) . lca is a rare and recently described vascular tumor of the spleen , first described by falk et al . , in 1991 . since the initial description , there have been scattered case reports and a few case series of lca . most of the cases of lca described in the literature have been composed of multiple nodules of varying sizes in the spleen ; solitary presentation is very rare . out of not more than 75 cases reported in english literature so far , only five cases were of a solitary mass.[46 ] lca is considered a benign neoplasm arising from the littoral cells lining the sinuses of the red pulp in normal splenic tissue . littoral cells possess morphologic and immunophenotypic features that reflect a dual endothelial and histiocytic potential of splenic sinus lining which are diagnostic traits for lca . morphological diagnosis is based on the presence of anastomosing vascular channels lined by tall endothelial cells , focal papillary fronds , and normal splenic sinuses at the periphery of the lesion . immunohistochemically , in contrast to normal sinus endothelium , which only expresses factor viii antigen and cd 8 positivity , lca neoplastic cells exhibit positive staining for both endothelial ( cd 31 and factor viii ) and histiocytic ( cd 68 and lysozyme ) antigens , and the complement receptors ( cd 21 and cd 163 ) . lca cells are negative for cd 8 , cd 34 , and s-100 protein antigens . lca has no particular gender or age predilection , although the median age in falk et al . 's original study was 49 years . the clinical presentation of lca ranges from completely asymptomatic and discovered incidentally , such as in our case , to a constellation of signs and symptoms such as abdominal pain , vague constitutional symptoms , splenomegaly , and hypersplenism . although first described as benign , a strong association has been shown between this neoplasm and a group of immunologic and oncologic entities . in approximately 36% of cases , lca was accompanied by immunodysregulation , which included crohn 's disease , metabolic disorders , and congenital abnormalities . in approximately 30% of the cases , lca was associated with various malignancies including colorectal , renal , hepatocellular , pancreatic , lung , lymphoma , myelodysplastic syndrome , and aplastic anemia . lca has also been shown to exhibit malignant potential , although this is extremely rare . the two subtypes of lca with malignant potential are described as littoral cell angiosarcoma and littoral cell hemangioendothelioma . histological evaluation reveals features consistent with lca histopathology , as well as abnormal architecture , nuclear atypia , and necrosis . the pathogenesis of lca remains unclear , but given its association with autoimmune disorders such as crohn 's disease , an immune system dysfunction has been postulated as a possible important pathologic mechanism . indeed , immune system dysfunction may explain the association of lca with other cancer types . these observations have prompted recommendations to closely evaluate and provide surveillance to patients with lca for the development of other malignancies . radiologically , lca may be evaluated by several imaging modalities such as ultrasound ( us ) , ct , mri , or nuclear medicine studies ( tc-99m - labeled rbc scintigraphy ) . the sonographic appearance of lca is variable and includes reports of mottled echotexture without discrete lesions , as well as findings of isoechoic , hypoechoic , and hyperechoic lesions . color flow doppler imaging may reveal the presence of central and peripheral vascularity that helps distinguish between lca and cavernous hemangioma of spleen . on noncontrast ct , lca appears as hypoattenuating masses ; given the vascular nature of these neoplasms , they tend to enhance homogeneously with contrast , particularly on delayed images . ct findings of surrounding capsule and internal calcification have not been described.[246 ] on mri , a minority of cases may be hypointense on both t1-weighted and t2-weighted scans because of the hemosiderin content of the tumor . however , no significant siderosis is seen in more than 50% of the cases and lesions tend to be hyperintense on the t2-weighted images . nuclear medicine studies with tc-99m - labeled rbc scintigraphy can be useful to differentiate splenic lesions from splenic hemangiomas . however , the radiologic features of lca are rarely diagnostic as many other splenic neoplasms such as hamartomas , hemangiomas , lymphomas , metastatic diseases , and infectious diseases exhibit similar imaging characteristics . symptomatic lca is often relieved by splenectomy , and given the association of lca with other malignancies and reported cases of malignant transformation , splenectomy is both diagnostic and therapeutic . in conclusion , lca is a recently described rare primary benign vascular neoplasm of the spleen that may be associated with other malignancies and may itself also have malignant potential . a majority of lcas are multinodular , although some may present as solitary lesion , as in our case .
summarypoly(adp - ribose ) polymerases ( parps ) ( also known as adp - ribosyl transferase d proteins ) modify acceptor proteins with adp - ribose modifications of varying length ( reviewed in refs 13 ) . parps regulate key stress response pathways , including dna damage repair and the cytoplasmic stress response2,3,4,5,6 . here , we show that parps also regulate the unfolded protein response ( upr ) of the endoplasmic reticulum ( er ) . human parp16/artd15 is a tail - anchored er transmembrane protein required for activation of the functionally related er stress sensors perk and ire1 during the upr . the third identified er stress sensor , atf6 , is not regulated by parp16 . similar to other parps that function during stress , parp16 enzymatic activity is up - regulated during er stress when it ( adp - ribosyl)ates itself , perk and ire1. ( adp - ribosyl)ation by parp16 is sufficient for activating perk and ire1 in the absence of er stress , and is required for perk and ire1 activation during the upr . modification of perk and ire1 by parp16 increases their kinase activities and the endonuclease activity of ire1. interestingly , the c - terminal luminal tail of parp16 is required for parp16 function during er stress , suggesting that it transduces stress signals to the cytoplasmic parp catalytic domain .
abstractimaging is important in the decision - making process of how to treat a lung tumour , which ideally should be a multi - disciplinary team decision . imaging is important during radiofrequency ablation ( rfa ) treatment with regard to optimal placement of the electrode , the immediate post - treatment criteria and very early detection of complications of the procedure . imaging is very important in the treatment follow - up . in lung rfa , as in many other interventional procedures , the traditional morphological imaging techniques to evaluate treatment response have difficulties and functional imaging techniques may potentially be more useful . however , larger studies showing this impact have not yet been performed .	although surgical resection is the preferred treatment at the early stages and confers the best outcome , only a quarter of patients are diagnosed in the early stages of non - small lung cancer ( nsclc ) . lobectomy is considered the standard of care in t1 n0 nsclc due to a low rate of local recurrence compared with sublobar resection . other studies , however , have demonstrated that the 5-year survival rate in the sublobar resection group was equal to lobectomy . the most common indication for sublobar resection in primary lung cancer is inadequate pulmonary reserve or comorbidities that contraindicate lobectomy . alternative local therapies such as radiofrequency ablation ( rfa ) and stereotactic radiation therapy may be attractive in this group of patients . the procedural mortality rate after rfa is 0.2% vs 1% after surgery and lung function does not deteriorate with repeated procedures when rfa is performed . several retrospective studies have demonstrated a survival benefit after resection of pulmonary metastases . in general , patients whose primary tumour is under control , with no extra pulmonary metastases , and who have a small pulmonary metastatic burden will benefit the most from surgical resection . currently , the major role of rfa in pulmonary metastases is the treatment of patients for whom surgical metastectomy would be ideal , but in whom comorbidities or technical issues render rfa more attractive . conventional treatment of inoperable or non - resectable lung tumours with systemic chemotherapy and conventional external beam radiation therapy has not been satisfactory in terms of survival outcomes . in general , tumours of up to 3 cm in diameter and located in the periphery of the lung are the ideal candidates for rfa . the rate of complete ablation in tumours larger than 3 cm in diameter has been shown to be poor in several studies . biologic tissues heated to greater than 50c for more than 5 min undergo coagulation necrosis . the area of coagulation is related to the strength of the radiofrequency energy , the current - carrying time , the diameter and shape of the electrode , and the composition of the surrounding tissues . both before , during and especially after rfa of a lung tumour , imaging is challenging , and this review focuses on some of these problems . proper pre - procedural staging is important in patients with nsclc or lung metastases as it will determine the best modality for treatment . staging should include chest and abdominal computed tomography ( ct ) together with positron emission tomography ( pet)/ct . the decision of how to treat a lung tumour should ideally be made by a multi - disciplinary team . rfa is performed with either a combination of local anaesthesia and conscious sedation or with general anaesthesia . the patient is placed supine or prone in the ct scanner and the shortest , most vertical path that avoids bullae , interlobular fissures or pulmonary vessels is chosen . tumour contact with a vessel of more than 3 mm in diameter is thought to create a heat sink effect that may render coagulation less successful . the relationship of the needle with the tumour must be assessed in 3 planes using image reconstructions . when expandable needles with multiple tines are used , it is important to assess the correct placement of the deployed tines before starting the ablation ( fig . figure 1rfa ablation is most often performed using ct as the modality of choice for guidance of placement of the rfa electrode . the relationship of the electrode needle with the tumour must be assessed in 3 planes : ( a ) axial , ( b ) sagittal , ( c ) coronal , and when needles with multiple tines are used , it is crucial to check the correct placement of the deployed tines before starting the ablation . rfa ablation is most often performed using ct as the modality of choice for guidance of placement of the rfa electrode . the relationship of the electrode needle with the tumour must be assessed in 3 planes : ( a ) axial , ( b ) sagittal , ( c ) coronal , and when needles with multiple tines are used , it is crucial to check the correct placement of the deployed tines before starting the ablation . in pathologic evaluation of microscopic tumour extension from nsclc , giraud et al . found that 95% of microscopic extension of primary nsclc would be encompassed in a margin of 8 mm for adenocarcinoma and 6 mm for squamous cell carcinoma . an rfa - induced area with ground glass is depicted in the rf ablated area immediately after the process and is easily seen on ct ( fig . the ground glass area around the tumour represents inflammation and should be considered a safety margin of pulmonary parenchyma that has been covered by treatment . so the target diameter of an ablation must ideally be at least 1 cm larger than the diameter of the tumour that undergoes treatment . proposed ground glass as an early indicator of treatment success after percutaneous rfa , and demonstrated that the point on the tumour surface where there is no ground glass margin is likely to be the site of future recurrence . figure 2post - procedure imaging ensuring that the tumour is surrounded by a circumferential area of ground glass of 1 cm is ideal , as areas of an ablated tumour with a small rim of ground glass are associated with future recurrent disease . in this case a sufficient area of ground glass is displayed . post - procedure imaging ensuring that the tumour is surrounded by a circumferential area of ground glass of 1 cm is ideal , as areas of an ablated tumour with a small rim of ground glass are associated with future recurrent disease . in this case tumour size and location has been linked to the rates of local tumour progression for lesions treated with rfa . a tumour size of less than 3 cm is associated with higher rates of complete tumour necrosis . after the procedure is completed a ct scan of the chest is obtained to detect pneumothorax , pleural fluid collection and haemorrhage . the procedure - related mortality rate is as low as 0.21% when 2905 ablations are considered . 2 ) was 28.3% , with 14.4% ( range 0.063.2% ) requiring aspiration or a chest drain . pleural effusions are often seen . however , the incidence of pleural effusions that need to be drained is in the order of 1 and 7% . other complications reported are haemoptysis , which usually does not require intervention , and infections . although the production of air micro - embolisms that pass from the pulmonary vein to the systemic circulation has been described , this phenomenon probably does not cause cerebral ischemia . accurately monitoring treatment response and early identification of residual or recurrent disease are critical for optimizing the effect of treatment . the crucial question for imaging follow - up is whether there is residual or recurrent viable tumour . response evaluation criteria in solid tumour ( recist ) is a widely accepted system that allows objective measurement of treatment response to chemotherapy . this system is based on changes in the diameter of the lesion either by ct or magnetic resonance imaging ( mri ) . when rfa of lung tumours is considered , the recist system is suboptimal to evaluate a response , as it can not differentiate viable from non - viable tumour or adjacent devitalized tissue . as the purpose of an appropriate rfa ablation of a lung tumour is to cause a coagulation necrosis larger than the initial lung tumour , contrast enhancement of the lesion can also be monitored , however hyperaemia and inflammation in the ablation zone may mask contrast enhancement of underlying residual tumour as reported by anderson et al . this group suggests that both size and enhancement of the ablated zone should be analysed to fully assess for residual tumour . as the tumour ideally becomes larger after a successful ablation , it has been recommended that a new baseline ct scan is performed 1 or 3 months after the ablation procedure . in fig . 3 it is obvious that the lung lesion in a successful ablation grows during the first months and afterwards decreases in size . one group found that the area of recurrent disease most often showed some degree of contrast enhancement . no stringent rules for the assessment of tumour response after rfa of a lung tumour exist . the features most commonly used to identify remaining viable tissue are tissue enhancement and nodular growth on serial images ( fig . 4 ) . figure 3the tumour size assessed by ct is larger than the volume of the tumour before ablation for a long time period . most authors recommend a 1- or a 3-months baseline scan to follow up the success of treatment . from this time point the tumour size on ct has to decrease if the ablation is successful as in this case . figure 4this figure illustrates a patient with recurrent disease 24 months after ablation shown by traditional morphological measurements suggesting growth of a part of the tumour ( c ) , perfusion ct ( f ) and pet / ct ( g ) and histopathology . ( d ) already indicates areas of perfusion probably indicative of recurrent disease , the amount of perfusion increased at the 18-month scan ( e ) and at the 24-month scan ( f ) . no tumour growth is seen on the ct scans at 12 months ( a ) and 18 months ( b ) . the perfusion ct ( d f ) is displayed as the 5-mm slice in which most perfusion is shown . the tumour size assessed by ct is larger than the volume of the tumour before ablation for a long time period . most authors recommend a 1- or a 3-months baseline scan to follow up the success of treatment . from this time point the tumour size on ct has to decrease if the ablation is successful as in this case . this figure illustrates a patient with recurrent disease 24 months after ablation shown by traditional morphological measurements suggesting growth of a part of the tumour ( c ) , perfusion ct ( f ) and pet / ct ( g ) and histopathology . the perfusion ct at 12 months ( d ) already indicates areas of perfusion probably indicative of recurrent disease , the amount of perfusion increased at the 18-month scan ( e ) and at the 24-month scan ( f ) . no tumour growth is seen on the ct scans at 12 months ( a ) and 18 months ( b ) . the perfusion ct ( d f ) is displayed as the 5-mm slice in which most perfusion is shown . in a large review , local recurrence was seen in 12.2% after a mean period of 13 months ( range 345 months ) . although no clear follow - up program exists , most authors perform contrast - enhanced ct at 1 , 3 , 6 , 9 , 12 months and then every 6 months following rfa . new imaging techniques may provide an opportunity for improved assessment of the post - therapy tumour bed . several retrospective and one prospective study evaluating pet / ct in the follow - up after rfa of lung tumours have been published . these studies in general suffer from a small number of patients , methodological problems due to retrospective design , a short and inhomogeneous time of follow - up and a suboptimal standard reference . however , it seems that ( 1 ) a large decrease in the standardized uptake value in the post - rfa fdg - pet , ( 2 ) a certain pattern of fdg uptake and ( 3 ) fdg uptake in the region around the original tumour indicating inflammation , are predictors of a successful ablation . to our knowledge , only one study has evaluated diffusion - weighted mri in a small number of patients in a retrospective design . diffusion - weighted mri performed 3 days after rfa showed reduced signal intensity and increased apparent diffusion coefficient values of the ablated lesions compared with pre - procedure tumour tissues . functional imaging probably will contribute in the work - up of treatment evaluation of lung tumour rfa ( fig . other image interpretation tools such as texture analysis may be useful in the follow - up of lung tumour ablation .
in microvillar photoreceptors , light stimulates the phospholipase c cascade and triggers an elevation of cytosolic ca2 + that is essential for the regulation of both visual excitation and sensory adaptation . in some organisms , influx through light - activated ion channels contributes to the ca2 + increase . in contrast , in other species , such as lima , ca2 + is initially only released from an intracellular pool , as the light - sensitive conductance is negligibly permeable to calcium ions . as a consequence , coping with sustained stimulation poses a challenge , requiring an alternative pathway for further calcium mobilization . we observed that after bright or prolonged illumination , the receptor potential of lima photoreceptors is followed by the gradual development of an after - depolarization that decays in 14 minutes . under voltage clamp , a graded , slow inward current ( islow ) can be reproducibly elicited by flashes that saturate the photocurrent , and can reach a peak amplitude in excess of 200 pa . islow obtains after replacing extracellular na+ with li+ , guanidinium , or n - methyl - d - glucamine , indicating that it does not reflect the activation of an electrogenic na / ca exchange mechanism . an increase in membrane conductance accompanies the slow current . islow is impervious to anion replacements and can be measured with extracellular ca2 + as the sole permeant species ; ba can substitute for ca2 + but mg2 + can not . a persistent ca2 + elevation parallels islow , when no further internal release takes place . thus , this slow current could contribute to sustained ca2 + mobilization and the concomitant regulation of the phototransduction machinery . although reminiscent of the classical store depletion operated calcium influx described in other cells , islow appears to diverge in some significant aspects , such as its large size and insensitivity to skf96365 and lanthanum ; therefore , it may reflect an alternative mechanism for prolonged increase of cytosolic calcium in photoreceptors .	light transduction in microvillar photoreceptors is a canonical instance of phosphoinositide signaling ( devary et al . , 1987 ) , and in all species that have been examined to date , the photoresponse is accompanied by a large elevation of intracellular free calcium ( brown and blinks , 1974 ; ranganathan et al . , 1994 ; calcium ions , long known for their pivotal role in light adaptation ( brown and lisman , 1975 ) , have also been implicated in the regulation of the visual excitation process ( bolsover and brown , 1985 ; payne et al . , 1986 ; werner et al . , 1992 ; shin et al . , 1993 ) , boosting the gain and speed of the light response ( payne and fein , 1986 ) . the detailed mechanisms of light signaling downstream of the hydrolysis of pip2 are yet to be fully elucidated , and there is evidence supporting both the insp3/ca branch of the cascade ( brown and rubin , 1984 ; fein et al . , 1984 ; payne et al . , 1986 ; shin et al . , 1993 ) , as well as dag or some metabolite thereof ( gomez and nasi , 1998 ; chyb et al . , 1999 ) ; nonetheless , the undisputed final effect is the opening of ionic channels in the plasma membrane , giving rise to an inward current . although in all microvillar photoreceptors the light - dependent conductance is cationic , there are significant species differences in ionic selectivity . in some organisms , like limulus , the light response is due to an influx of na ions , with little or no detectable contribution by ca ( millecchia and mauro , 1969 ; brown and mote , 1974 ) . in contrast , in others , like balanus and drosophila , calcium is a major charge carrier of the photocurrent ( brown et al . , 1971 ; hardie , 1991 ) . not surprisingly , there are concomitant differences in the source of the ca mobilized by light . in limulus and apis , the light - induced increase of cytosolic ca is entirely accounted for by release from internal stores ( brown and blinks , 1974 ; baumann et al . , 1991 ) triggered by stimulation of insp3 receptors ( brown and rubin , 1984 ) , which are confined to the light - sensitive lobe of the cell ( payne and fein , 1986 ) . in balanus and drosophila , instead , this is attributable primarily to influx , as removal of extracellular calcium greatly depresses the ca elevation ( brown and blinks , 1974 ; ranganathan et al . , 1994 ) . because of the ubiquitous role of calcium in the regulation of light responsiveness , intense and/or prolonged photostimulation poses a challenge for those cells possessing light - activated channels that are poorly permeable to this ion , as the internal pool of calcium is inevitably limited . we addressed this problem by examining microvillar photoreceptors of the marine mollusk lima scabra because an extensive electrophysiological characterization had lead to the conclusion that there is minimal , if any , permeation of calcium through the light - sensitive channels ( gomez and nasi , 1996 ) . through the combined use of patch clamp recording and calcium fluorescence measurements , we identified a slow ca - dependent inward current that only becomes manifest under a regimen of intense photostimulation when further internal release can not be sustained . this novel conductance fulfills the requirements for mediating the persistent elevation of intracellular calcium necessary to regulate the light - transducing machinery under such conditions . were dissected under dim red illumination and enzymatically dispersed as described previously ( nasi , 1991a ) . the resulting suspension of dissociated microvillar photoreceptors was plated in a recording flow chamber mounted on the stage of an inverted microscope . the chamber was continuously superfused ( 1 ml / min ) with artificial sea water ( asw ) ; a system of manifolds permitted the exchange of the superfusate . for rapid local solution changes , a puffer micropipette was positioned in the vicinity of the cell and could pressure - eject a stream of test solution upon activation of a solenoid - operated valve . whole cell patch clamp recordings were performed as described previously ( nasi , 1991b ) . signals were low - pass filtered at 100 hz for slowly changing currents and at 1,000 hz for the rapid light - evoked current using a bessel four - pole filter . records were digitized online at a sampling rate of 200 hz and 3 khz , respectively . all cell manipulations were performed under dim near - infrared illumination ( long - pass filter , > 715 nm ; andover ) . light flashes were generated by a conventional optical stimulator delivering small spots of light ( 200 m in diameter ) . an in vivo calibration was used to quantify light intensity in terms of effective photons s cm at 500 nm , as measured with a radiometer ( united detector technology ) . voltage and light stimuli were applied by a microprocessor - controlled programmable stimulator ( stim 6 ; ionoptix ) . changes in cytosolic ca were detected using visible light fluorescent indicators ( calcium green 5n , fluo 4 , and oregon green 2 ; invitrogen ) . the potassium salt of the probe was dissolved in the intracellular solution filling the patch electrode at a final concentration of 60100 m . excitation light provided by a 75-w arc lamp ( xenon ; pti ) was filtered by a dichroic reflector to reject wavelengths longer than 670 nm ( omega optical ) , and by an interference filter ( 480 nm , 40-nm bandwidth ; chroma technology corp . ) . the beam was brought to the epi - illumination port of the microscope via a liquid light guide ( oriel corporation ) . emission light collected by a 100 , 1.3 numerical aperture oil - immersion objective ( nikon ) was filtered sequentially by an additional dichroic ( < 610 nm ) and by a 535-nm interference filter ( 50-nm bandwidth ; all from chroma technology corp . ) . an adjustable mask ( nikon ) located at a conjugated image plane was positioned under infrared visualization to restrict the collected light to a rectangular region of interest ( the light - sensitive lobe of the cell , unless otherwise stated ) and thus minimize background light . the fluorescence signal was detected by a photomultiplier tube ( model r4220 pha ; hamamatsu photonics ) operated at 800 v in photon - counting mode , using a window discriminator and a rate meter ( f-100 t and prm-100 ; advanced research instruments ) . an analogue voltage proportional to the counts accumulated in bins of programmable duration was fed to the a / d interface of the computer . asw contained ( in mm ) : 480 nacl , 10 kcl , 10 cacl2 , 49 mgcl2 , 10 hepes , and 5.5 glucose , ph 7.8 ( naoh ) . in na - free sea water , sodium was replaced by nmdg , lithium , or guanidinium . in ( nominally ) calcium - free asw , calcium was replaced iso - osmotically with magnesium , without adding chelators . in low - chloride asw , [ cl ] was reduced by 500 mm , replacing nacl and cacl2 with na- and ca - gluconate , respectively . to test the permeation of different divalent cations , the extracellular solution contained 60 mm calcium , barium , or magnesium , 490 mm nmdg cl , and 10 mm hepes / tris . the standard intracellular solution used to fill whole cell micropipettes contained 200 mm k - glutamate , 100 mm kcl , 22 mm nacl , 5 mm mg atp , 10 mm hepes , 1 mm egta , 100 m gtp , and 300 mm sucrose , ph 7.3 . in some instances , intracellular sodium was omitted to forestall the possibility of reverse operation of the na / ca exchanger . to increase the internal solution with elevated ca had a similar composition , except that 0.5 mm cacl2 was added to it with 1 mm tetrafluoro - bapta ( instead of egta ; invitrogen ) , yielding an estimated free calcium concentration of 50 m . for perforated patch recording , the solution used to backfill the electrode contained 0.1 mg / ml nystatin , and membrane perforation was monitored by the gradual appearance of capacitative transients elicited by voltage steps . we also used -escin ( fan and palade , 1998 ) , which produces more sizable pores but still prevents the washout of molecules > 10 kd . asw contained ( in mm ) : 480 nacl , 10 kcl , 10 cacl2 , 49 mgcl2 , 10 hepes , and 5.5 glucose , ph 7.8 ( naoh ) . in na - free sea water , calcium - free asw , calcium was replaced iso - osmotically with magnesium , without adding chelators . in low - chloride asw , [ cl ] was reduced by 500 mm , replacing nacl and cacl2 with na- and ca - gluconate , respectively . to test the permeation of different divalent cations , the extracellular solution contained 60 mm calcium , barium , or magnesium , 490 mm nmdg cl , and 10 mm hepes / tris . the standard intracellular solution used to fill whole cell micropipettes contained 200 mm k - glutamate , 100 mm kcl , 22 mm nacl , 5 mm mg atp , 10 mm hepes , 1 mm egta , 100 m gtp , and 300 mm sucrose , ph 7.3 . in some instances , intracellular sodium was omitted to forestall the possibility of reverse operation of the na / ca exchanger . to increase the internal solution with elevated ca had a similar composition , except that 0.5 mm cacl2 was added to it with 1 mm tetrafluoro - bapta ( instead of egta ; invitrogen ) , yielding an estimated free calcium concentration of 50 m . for perforated patch recording , the solution used to backfill the electrode contained 0.1 mg / ml nystatin , and membrane perforation was monitored by the gradual appearance of capacitative transients elicited by voltage steps . we also used -escin ( fan and palade , 1998 ) , which produces more sizable pores but still prevents the washout of molecules > 10 kd . the receptor potential of lima rhabdomeric photoreceptors has a complex waveform ( nasi , 1991a ) , owing to the presence of two distinct conductances that are directly dependent on light stimulation ( nasi , 1991c ) as well as voltage- and calcium - dependent ion channels that shape its time course ( nasi , 1991b ) and endow these cells with the ability to generate propagating action potentials ( mpitsos , 1973 ) . as the intensity of stimulating flashes is raised to high levels , an additional feature appears : seconds after the receptor potential ( generally accompanied by one or more action potentials ) , the membrane voltage gradually depolarizes by 1020 mv ( fig . the voltage remains depolarized for 13 min , and then slowly returns to the dark resting level . b illustrates the prolonged time course of this phenomenon , recorded in different cells . during we used voltage clamp recording to investigate this long - lasting electrical response evoked by light . 1 c demonstrates that a bright flash delivered from a holding voltage near the cell 's resting potential elicits a slow inward current ( henceforth referred to as islow ) , with temporal characteristics that parallel those of the aforementioned after - depolarization ( the early , truncated downward transient is the primary photocurrent , highly compressed at this time scale ) . islow amplitudes typically vary between 50 and 250 pa ( 113 37 pa sd in 22 cells tested under the same stimulation conditions ) . the phenomenon is robust and reproducible ( n > 100 ) , although there is across - cells variability not only in the peak amplitude of the slow light - induced inward current , but also in its time course ; nonetheless , its duration is consistently at least two orders of magnitude greater than that of the primary photocurrent . islow can be triggered repeatedly in a given cell , and its features are reproducible , provided that the stimulation is not too intense ( fig . 1 e ) ; in fact , this slower response often proved to be more robust than the photocurrent itself . to rule out the possibility that islow may artifactually result from the perturbation of the intracellular milieu or the loss of cell constituents during dialysis by the patch pipette , we resorted to the minimally invasive perforated patch technique ( horn and marty , 1988 ) . f shows a current trace recorded after perforation of the membrane patch with nystatin ; islow is indistinguishable from the currents recorded by means of the conventional whole cell patch clamp technique ( n = 4 ) . ( a ) current clamp recording in a cell stimulated with a bright flash of light ( 5.4 10 photons cm s ) . after the complex - shaped receptor potential and a train of action potentials , membrane voltage drifted in the depolarizing direction by 15 mv . ( b ) recordings in three different cells , shown at a more compressed time scale , to illustrate the prolonged time course of the after - depolarization . ( c ) a similar photostimulation applied to a different cell under whole cell voltage clamp , internally dialyzed with standard intracellular solution . a islow started to develop several seconds after the photocurrent ( which was truncated , as its amplitude of several na would fall off - scale at this gain ) . ( d ) slow after - current in three different cells to illustrate the variability in amplitude and time course . ( e ) repetitive activation of islow in the same cell , exposed to a flash attenuated by 0.6 log compared with the trace in c and d. ( f ) islow obtains in photoreceptors voltage clamped by the perforated patch technique . the two examples show the current evoked by a 100-ms flash ( 1.4 10 photons cm s ) in photoreceptor cells in which access to the cytoplasmic compartment was attained by adding the pore - forming agent nystatin to the pipette filling solution , or -escin . in all these examples , a striking feature of islow is that with dim or moderate illumination it is virtually undetectable and only becomes conspicuous at light intensities in the saturating range for the photocurrent . from that point on , its amplitude is graded with stimulus intensity , while the size of the photocurrent remains essentially unchanged . 2 b illustrates instead a typical intensity series for the photocurrent recorded in a different cell and displayed at a lower gain and faster time scale . it can be appreciated that islow only appears with flash attenuation ( 2.7 log ) at which the photocurrent has already attained its maximal amplitude , and continues to increase monotonically over at least 2 log units ( n = 2 ) . in fig . response relation was plotted for both the regular photocurrent and for islow ( n = 3 in each case ) to highlight the profound difference in light intensity dependency ( 3 log units ) . 2 d shows that the slow current also obtains with prolonged illumination of more moderate intensity , such as it may prevail in shallow waters on a sunny day , indicating that its occurrence is not alien to the conditions that the organism is likely to encounter in its environment , which is sub - tidal to a few meters of depth ( where total incident light flux can be in excess of 10 photons cm s ) . ( a ) islows evoked in a cell stimulated with 100-ms flashes of increasing intensity , as indicated by the attenuation factor on the right . ( b ) intensity series for the photocurrent in a different photoreceptor displayed at a faster time scale . the amplitude of the response reaches saturation at a stimulus intensity below the threshold for the slow current . ( c ) plot of the peak amplitude of the photocurrent ( open symbols ) and that of the slow aftercurrent ( filled symbols ) averaged for three cells in each condition . ( d ) the slow current is also elicited with more modest stimulus intensities , provided that their duration is extended . the panel shows the response to a step of light attenuated by 1.8 log but maintained for the duration of the recording . holding potential of 50 mv in all panels , unattenuated light intensity of 19 10 photons cm s. illumination of lima microvillar photoreceptors produces a large elevation of cytosolic ca . 3 a ( left ) illustrates an example of simultaneous measurement of membrane current and ca fluorescence in a voltage - clamped cell . a low - affinity indicator , calcium green 5n , was loaded by perfusion via the patch pipette at a final concentration of 63 m . activation of the epi - illumination beam triggered two events : ( 1 ) a vigorous inward photocurrent ( fig . 3 a , top trace ) , and ( 2 ) an instantaneous transition of the photomultiplier output signal ( reflecting the basal fluorescence ) , followed by a large ca rise concomitant with the onset of the photocurrent ( middle trace ) . the fluorescence reached a peak within 70 ms from the beginning of the light and then decayed . 3 a ( right ) demonstrates that light - induced ca transients survive removal of extracellular calcium ( the apparently more rapid decay in calcium - free medium was not consistently observed , and likely reflects cell - to - cell variability ) . further support for this contention is provided by spatially resolved measurements of the activation kinetics of the calcium signal , relative to that of the electrical response . to this end , the optical recording was circumscribed to a small window , 3 3 m , the position of which was manipulated under infrared light visualization . 3 c , which shows a nomarski micrograph of a photoreceptor with the positions of the optical mask indicated by squares . in fig . 3 b , membrane current ( inverted for display purposes ) and ca fluorescence traces were normalized and overlaid . when the fluorescence signal was recorded from the microvillar lobe ( rhabdomere ) , the rise in calcium slightly preceded the photocurrent onset . when the window was positioned onto the cell body ( soma ) , the ca fluorescence rise significantly lagged behind the membrane current ( notice the different time scales ) . in fig . 3 d , pooled data for a group of cells are shown in a bar graph . in the microvillar lobe , the calcium signal led the light - evoked current by 1.1 0.95 ms ( sd ; n = 14 ) , whereas when the fluorescence was recorded from the middle of the somatic lobe , it lagged by an average of 16.4 2.1 ms ( n = 5 ) . the amplitude of the ca fluorescence signal was also different , being significantly larger in the microvillar lobe than in the soma ( not depicted ) . in conclusion , ca elevation originates in the photosensitive region of the photoreceptor , it precedes the opening of light - dependent ion channels , and it does not require extracellular calcium ; as a consequence , it must implicate light - induced intracellular release . light stimulation releases calcium from intracellular stores in the microvillar lobe of lima photoreceptors . ( a ) simultaneous recording of membrane current under voltage clamp ( top traces ) and fluorescence of the indicator calcium green 5n , which was dialyzed through the patch pipette at a concentration of 63 m ( bottom traces ) . a large increase in fluorescence above the initial level occurred shortly after activating the epi - fluorescence beam , coincident with the beginning of the photocurrent . a similar ca transient was observed in a cell superfused for 8 min with nominally calcium - free solution ( right ) . ( b ) the ca transient originates in the microvillar lobe and precedes the opening of the light - dependent channels . the fluorescence was recorded from a small window positioned either on the microvillar lobe ( rhabdomere ) or on the cell body ( soma ) , as indicated in c. the signal from the rhabdomere slightly preceded the membrane current ( inverted for display purposes and normalized ) , whereas that from the cell body lagged behind the electrical response by many milliseconds . ( d ) pooled data of the temporal shift between current and fluorescence , averaged for several cells tested in the two arrangements . after termination of photostimulation , the large accumulation of cytosolic calcium is rapidly reduced , as determined by a double - stimulus protocol . in such measurements , the excitation light is presented briefly twice ( 80100 ms each time , sufficient to encompass the peak of the light - induced ca increase ) . the duration of the intervening interval is varied across cells . as shown in fig . 4 a , the first flash ( left ) evoked a conspicuous rise in fluorescence over the initial baseline level ( marked by a dotted line ) . in these and all subsequent fluorescence measurements , the optical mask restricted light collection to the whole light 4 a , right ) , no additional light - induced calcium increase was observed ; however , the level of fluorescence remained significantly elevated , but not as high as the peak attained with the first flash . by comparing the relative fluorescence ( f / f ) at the peak of the first response with that in the second flash as the interval is varied , one can determine the time course of the fall in ca fluorescence after the light is turned off . 4 b shows that in 3 s the residual calcium signal had decayed to 58 15% ( n = 6 ) , and in 5 s it decreased to 30 18% ( n = 3 ) . ( a ) fluorescence was measured in response to pairs of brief flashes ( 90 ms ) spaced 3 s apart . the first light evoked a large increase in ca fluorescence above the basal level . at the time the second flash was presented , fluorescence remained significantly elevated , and no further increase was elicited . ( b ) average amplitude of the fluorescence signal during the second stimulus , normalized with respect of the initial peak amplitude , as the interval separating the two flashes was increased from 3 to 5 s. error bars indicate standard deviation . one mechanism that has been implicated in restoring basal ca levels in microvillar photoreceptors is the na / ca exchanger , which , owing to the stoichiometry of the countertransport process ( na / ca > we investigated whether islow may reflect the activation of an electrogenic na / ca exchanger . considering the sluggish onset of this current ( in contrast with the rapid elevation of [ ca]i ) , this possibility would require that the exchanger be spatially segregated with respect to the source of calcium . we tested the effect of removing extracellular sodium to prevent forward operation of the exchanger . substitution with li ( n = 9 ) resulted in a slow current with normal characteristics , indistinguishable from control measurements . n = 8) ; compared with lithium , the only salient difference was the reduction in the amplitude of the initial light response , as expected from the fact that guanidinium and nmdg permeate poorly through the light - sensitive channels , whereas lithium can substitute for na as the carrier of the inward photocurrent ( gomez and nasi , 1996 ) . islow is not due to a na / ca exchanger . to evaluate the possibility that the slow current may reflect the activity of an electrogenic na / ca exchanger triggered by the light - evoked increase in cytosolic ca , cells were stimulated with bright flashes while superfused with solution containing no sodium . the slow current was still observed , regardless of the replacement of extracellular sodium . with lithium , which permeates readily through the light - dependent ion channels , a large photocurrent ( truncated in the figure ) , the initial inward current was severely reduced ( < 100 pa ) , as the photoconductance is poorly permeable to these ions ; nonetheless , the subsequent slow current developed normally , both in terms of time course and amplitude . light intensity of 1.6 10 photons cm s ; holding potential of 60 mv . one can conclude that islow is not the electrical manifestation of the sodium - dependent extrusion of the calcium released upon photostimulation . rather , it appears to correlate with a sustained increase in cytosolic calcium for the following reasons : the data in fig . 4 indicate that after a brief flash of very high intensity , such as the epi - fluorescence excitation beam , subsequent flashes do not release additional ca ( as indicated by the flat time course of the fluorescence trace during the second flash ) . because the clearance of the calcium load occurs with an initial time constant of < 5 s , one would expect [ ca]i to return to baseline levels within 20 s or less . 6 a , which shows a measurement of membrane current and ca fluorescence protracted over a 1-min period . because we were interested in the lower amplitude plateau elevation of ca , a higher affinity indicator was chosen for this task ( fluo 4 ) . to minimize bleaching of the calcium indicator , the epi - fluorescence illumination was not continuous but pulsed , delivering 80100-ms flashes every 3 s , and the fluorescence was sampled concomitantly with the membrane currents during such episodes . four readings were taken before initiating the train of flashes ( for the fluorescence channel , these represent dark counts ) . with the first flash , the fluorescence signal displayed a sharp rise ( both the foot level of basal fluorescence and the peak are plotted in the graph , represented by the open and the filled squares , respectively ) , and then decayed on subsequent trials , stabilizing at a level that was higher than the initial fluorescence . concomitantly , several seconds after the first flash , islow started to develop , reaching an amplitude of 186 pa . on average , the plateau fluorescence at the end of the recording was higher than the basal level by 20 8.5% ( sem ; n = 5 ) . because even with the pulsed protocol the cumulative light exposure was not inconsequential , we gauged whether significant bleaching of the probe may have occurred . to eliminate the confounding due to the continuous replenishment via dialysis , the patch pipette was withdrawn after loading the dye into the photoreceptor , as depicted schematically in fig . 6 b. the epi - illumination beam was turned on for 5 s , producing the characteristic ca fluorescence increase ( fig . the duration of the rest period was chosen to allow for the ca elevation to clear and for islow to die out . 6 d , the foot of the optical signal on the second stimulation ( shown also on an expanded time scale on the right ) was not significantly depressed with respect to the initial basal fluorescence level , suggesting that bleaching of the ca dye was marginal ( n = 3 ) ; therefore , the plateau ca fluorescence in fig . the persistent , though modest , elevation of cytosolic calcium , in spite of the lack of any further detectable photo - induced release after the initial flash , implies that some source of ca must be active . the fact that the lingering ca fluorescence paralleled the slow current suggests that the latter may be associated with ca influx . ( a ) membrane current was measured concomitantly with ca fluorescence of the rhabdomeric lobe in a cell loaded with the indicator fluo 4 ( 83 m ) . recordings were made every 3 s ; after four baseline measurements , excitation flashes ( 90 ms in duration ) were delivered repetitively . the bottom graph plots the fluorescence signal , which decayed partially after the initial large increase , but remained significantly elevated with respect to the basal level . the open square and dotted line mark the basal fluorescence level during the first flash , and the highest point represents the peak fluorescence level attained during the same flash ( in subsequent flashes the trace was flat during the light , as there was no further release ; therefore , the foot vs. peak ( b ) to estimate whether dye bleaching might contribute significantly to the decay of the fluorescence signal , owing to the extended light exposure , photoreceptor cells were loaded with the ca indicator for 4 min and the pipette was withdrawn to prevent any further dye exchange . ( c ) the epi - fluorescence beam was activated for 5 s , evoking a large optical signal that decayed to a plateau ( basal fluorescence is indicated by the dotted line ) . ( d ) after 10 min , the excitation light was turned on again , and the basal fluorescence was found not to be noticeably depressed with respect to the initial level ( i.e. , immediately after the first opening of the shutter ) . the initial portion of both fluorescence traces is displayed ( on the left they were displaced vertically for clarity ; the arrowheads mark the basal fluorescence levels ) . the slower rise of the light - induced ca increase on the second trial indicates physiological rundown . on the right , the same traces are superimposed and shown on an expanded time scale . because no replenishment of dye was possible under these conditions , the results indicate that a 5-s exposure to the excitation light did not result in significant bleaching of the calcium probe . measurements of input resistance of the cell after delivery of a bright flash reveal that an increase in membrane conductance is responsible for islow . in fig . 7 a , a photoreceptor was voltage clamped at 60 mv , but the holding potential was subjected to a repetitive square - wave perturbation ( 10 mv amplitude , 10 hz , 50% duty cycle ) . stimulation with a bright flash triggered the slow current ; concomitantly , the amplitude of the current excursions produced by the voltage steps grew ( fig . 7 a , insets ) , reporting an increase in membrane conductance . with saturating flashes , the conductance change was approximately sixfold , from 2.8 0.7 ns ( sem ) to 13.4 3.7 ns ( n = 13 ) , for an average peak amplitude of islow of 255 39.6 pa . these values would predict an average peak amplitude of the prolonged depolarization on the order of 19 mv , consistent with the values measured under current clamp ( see fig . 1 b ) . ( a ) the photoreceptor input resistance was continuously monitored by applying a repetitive rectangular command step , 10 mv in amplitude , superimposed upon the steady holding potential ( 60 mv ) . after the flash , the size of the current steps gradually increased ( insets ) concomitantly with the development of the slow current , indicating a reduction in membrane resistance . ( b ) to assess the permeation of individual divalent cations , photoreceptors were tested in an extracellular solution containing either 60 mm ca or 60 mm mg as the sole divalent species . the internal solution contained no sodium to prevent calcium loading via reverse operation of the na / ca exchanger , and the holding potential was set at 50 mv . a robust islow was observed in the presence of ca ( top trace ) , whereas it was entirely absent with mg ( bottom trace ) . light intensity of 3.2 10 photons cm s. ( c ) islow does not invert with membrane depolarization to + 40 mv , whereas the primary photocurrent does . second , the slow current is cationic and carried at least in part by calcium . the participation of chloride was dismissed upon observing that a reduction in extracellular cl concentration from 608 to 108 mm by equi - molar replacement with gluconate produced no significant effects on the slow current ( not depicted ; n = 3 ) . likewise , the data shown in fig . 5 rule out a unique contribution of na . the permeation of calcium was assessed by superfusing the cell with solutions containing only one divalent cation at a time . with 60 mm cacl2 , in contrast , if magnesium was the only divalent cation in the extracellular solution , the slow current was virtually absent ( fig . 7 the internal solution contained no sodium to prevent calcium loading via reverse operation of the na / ca exchanger . 7 c shows that the slow current remains inwardly directed at holding potentials up to + 40 mv ( n = 3 ) . larger depolarizations proved deleterious to the cells ( even when the recording window was shortened to 1 min , like in fig . 7 c ) , so we were unable to revert islow ; moreover , the presence of voltage- and calcium - activated currents of considerable amplitude ( several na ) ( nasi , 1991b ) would likely defeat any attempt to obtain an accurate estimate of its vrev . the properties of islow described above are reminiscent of those of store - operated currents that have been described in a wide variety of cells that use insp3-mediated ca signaling . an important experimental tool for determining that they depend on the filling state of the internal calcium stores per se ( rather than messengers generated by the plc pathway ) is thapsigargin , an inhibitor of the calcium pump of the endoplasmic reticulum ( serca ) . thapsigargin can slowly empty calcium stores and trigger a sustained subsequent influx of calcium in many cell types . we found thapsigargin ( 400 nm ) to be minimally effective in altering membrane current in voltage - clamped lima photoreceptors . 8 a shows the largest effect we observed in response to local application via a puffer pipette . another cell produced an even smaller current ( < 10 pa ) , and a third did not respond at all . monitoring the membrane current for several additional minutes beyond the interval shown in fig . 8 b ) with an amplitude not significantly different from that of control cells ( 107 27.2 pa sd ; n = 3 ) ; however , as shown by the inset of fig . 8 b , the primary photocurrent was greatly depressed , attaining a size of only a few hundred pa ( compared with several na in control cells ; see fig . 2 b ) . to further assess whether the thapsigargin treatment was effective , the light - evoked elevation of intracellular calcium was monitored with fluorescent indicators . 8 c shows the fluorescence in control cells and in cells treated with 400 nm thapsigargin . the drug nearly abolished the large , rapid elevation of ca that typically begins 30 ms after opening the epi - illumination shutter . the data averaged from six cells , three in each condition , are summarized by the bar graph in fig . 9 a shows that puffer application of 50 m sfk96365 during islow failed to alter its time course ( n = 4 ) . application by continuous bath superfusion was also ineffective ( n = 2 ) . lanthanum has also been reported to block capacitative ca influx at submillimolar concentrations in several cell types . fig . 9 b shows that during extracellular application of 1 mm la by continuous superfusion of the entire recording chamber , a very conspicuous islow could still be elicited ; another cell generated a slow current of 148 pa . to assess whether islow had suffered any attenuation at all , we resorted to a more sensitive within - cell comparison and found that , once the slow current had fully developed , brief local application of lanthanum produced only a marginal inhibitory action ( 15% ; n = 4 ; not depicted ) . in several key aspects , therefore , islow departs from the features commonly encountered in store - operated currents . ( a ) application of 400 nm thapsigargin in the dark by local superfusion ( indicated by the horizontal line beneath the trace ) only produced a very small change in membrane current . ( b ) islow evoked in a thapsigargin - treated cell has a normal amplitude and time course . ( inset ) photocurrent measured in response to bright flashes , showing that the light response is greatly attenuated . cells were loaded with 83 m oregon green2 by dialysis . compared with the pronounced increase in ca fluorescence consistently recorded in control cells ( left traces ) , in thapsigargin - treated cells ( 400 nm ) ( d ) average light - induced increase in ca fluorescence for three control cells and three cells exposed to thapsigargin . ( a ) 50 m skf96365 was continuously pressure - ejected by a puffer pipette , beginning shortly after the slow current had peaked ( horizontal line ) . there was no discernible alteration in the time course of the current , indicating that islow was not suppressed . vh = 50 mv ; light intensity of 28.6 10 photons cm s ) . ( b ) a large - amplitude islow elicited in the presence of 1 mm la ( applied by extensive bath superfusion before applying the light stimulus ) . vh = 60 mv ; light intensity of 2.39 10 photons cm s ) . in addition to capacitative ca entry , plc - triggered pathways for calcium influx that are not dependent on the filling state of the intracellular calcium stores have also been described . arachidonic acid can activate such noncapacitative ca influx in some cases ( shuttleworth , 1996 ) . we tested stimulation of lima microvillar photoreceptors with arachidonic acid either applied by local superfusion ( 1050 m ; n = 9 ) or by internal dialysis ( 10 m ; n = 3 ) . such treatment , however , failed to elicit any discernible inward current . we also examined the possibility that calcium itself may be a triggering stimulus for islow . to that end , we dialyzed elevated calcium into the cells ( 50 m ) but did not observe the spontaneous development of an inward current in the dark . moreover , high intracellular calcium depressed islow rather than potentiating it . compared with control photoreceptors dialyzed with the standard internal solution , 50 m [ ca]i reduced approximately by half the amplitude of islow ( 119 54 pa ; n = 9 ) . thus , it seems unlikely that the rise in cytosolic ca levels directly controls islow , a contention that would also be difficult to reconcile with the time course of this membrane current , which develops over many seconds , long after the increase in [ ca]i has substantially decayed . the ubiquitous phosphoinositide signaling pathway ( for review see berridge and irvine , 1989 ) underlies visual transduction in microvillar photoreceptors of invertebrates ( devary et al . , 1987 ) . according to the canonical scheme , the ca elevation that accompanies the photoresponse is initiated by release from internal insp3-sensitive stores . owing to the finite capacity of the calcium - containing organelles , such release can not proceed indefinitely when a cell is challenged with strong or sustained stimulation . if the light - activated channels are permeable to calcium ( brown et al . , 1971 ; hardie , 1991 ) , ca signaling can be protracted simply via influx through the same ionic pathway that underlies the receptor potential . however , in several microvillar photoreceptors , the light - dependent conductance does not allow significant ca fluxes , an impasse that calls for ancillary mechanisms to aid the prolongation of the calcium response . the present study was inspired by the observation that lima microvillar photoreceptors exposed to strong photostimulation produce a receptor potential that is consistently followed by an after - depolarization lasting for > 1 min ( nasi , 1991a ) ; interestingly , ca does not permeate significantly through the light - dependent conductance of lima photoreceptors ( gomez and nasi , 1996 ) . a similar slow after - depolarization had been noted in apis ( baumann and hadjilazaro , 1972 ) . our data demonstrate the presence of a separate , slowly activating ionic mechanism , islow , which is only triggered with brief , saturating flashes , or with prolonged light stimulation , and is permeable to calcium ions . measurements with fluorescent calcium probes show that , concomitantly with islow , calcium is maintained at an elevated plateau for tens of seconds . from the time constant of the decay of the initial light - evoked transient of ca release , one would not anticipate such lingering calcium increase , which must therefore be attributed to islow . consequently , this conductance is ideally suited to help in the ca - dependent regulation of the light transduction machinery under conditions when the process of internal release ceases . this differs from a mechanism previously described in the drone retina , in which the recovery of photo - induced depletion of sequestered calcium was aided by the application of sustained , dim illumination ( ziegler and walz , 1990 ) . coping with the need to mobilize calcium beyond the capacity of the intracellular stores is , of course , a challenge not unique to photoreceptors . in many other cells that rely on phosphoinositide signaling , after the initial ca release subsides a persistent influx of calcium through the plasma membrane is often evoked , which has been dubbed capacitative ca entry ( for review see putney , 1990 ; parekh and penner , 1997 ) . additionally , ca influx can also occur through a pathway dependent on receptor activation , but not necessarily on the filling state of the stores ( receptor - operated current ; for review see bolotina , 2008 ) . in both cases , the exact mechanism that controls , 2001 ; venkatachalam et al . , 2002 ; prakriya and lewis , 2003 ) , but it may involve a diffusible internal messenger ( parekh et al . , 1993 ; randriamampita and tsien , 1993 ) , direct conformational coupling between proteins ( luik et al . , 2006 ) , or the exocytotic fusion of channel - containing vesicles with the plasmalemma ( yao et al . , 1999 ) . the molecular search for some of the players has led to the identification of a candidate molecule for the role of er luminal ca sensor , stim1 ( roos et al . , 2005 ) , and a putative subunit of the calcium channel , orai1 ( feske et al . , 2006 ) . there are obvious similarities between islow and depletion - activated currents , including the striking resemblance in time course and the conditions of stimulation under which it is elicited . however , other features of islow are surprising , such as its amplitude , which , although 20-fold smaller than the photocurrent , is still quite considerable . in contrast , in a variety of other cells the currents associated with the late ca influx proved very difficult to measure electrophysiologically because of their minute size ( typically only a few pa ; e.g. , hoth and penner , 1992 ; bahnson et al . , 1993 ) . two considerations could explain the large amplitude of islow in lima : ( 1 ) calcium transients in microvillar photoreceptors are the largest known , reaching amplitudes of tens of m ( brown et al . , 1977 ; , 1994 ; ukhanov et al . , 1995 ) , and lima is no exception . although an absolute quantification is not possible without ratiometric measurements , large signals were obtained with a low - affinity dye ( ca green 5n ; kd 14 m ) ; moreover , signals recorded with higher affinity indicators such as fluo 4 and calcium green 2 ( kd 0.345 and 0.55 m , respectively ) had slower fall kinetics , suggestive of dye saturation ( not depicted ) . notice that all kd values quoted refer to the manufacturer specifications , measured in 100 mm kcl , but for all calcium indicators the affinity is known to decrease significantly in vivo and at higher values of ionic strength ( the ionic strength of asw is 1.35 ; thomas et al . , 2000 ) . to the extent that a sizable fraction of such a massive load of released calcium is rapidly extruded by a na / ca exchanger ( minke and armon , 1984 ; o'day and gray - keller , 1989 ) , the task of subsequently reestablishing homeostasis may require a correspondingly hefty pathway of ca influx . ( 2 ) the heavily invaginated rhabdomere provides abundant membrane area to accommodate a large number of transporting proteins ( as gauged from the capacitance , which , at 70 pf , is severalfold larger than that of other cells of similar dimensions ) ( nasi , 1991a ) . this would make lima photoreceptors an appealing model for the biophysical characterization of the slow ca influx , offering , in a native context , the size and reproducibility that in other cells have only been obtained by overexpression of stim and orai ( mercer et al . , 2006 ; peinelt et al . , 2006 ; soboloff et al . , 2006 ) however , differences between islow and the classical capacitative calcium entry must not be disregarded . one is that thapsigargin , a serca inhibitor widely used to empty internal calcium stores and capable of mimicking physiologically induced soce , only elicited a very modest current . independent evidence demonstrated that under those conditions the cells ' ability to release calcium was severely compromised , suggesting that thapsigargin had indeed depleted calcium stores . an equally salient discrepancy is the lack of suppression of islow by lanthanum and by skf96365 , two agents known to block capacitative ca entry . at this stage , therefore , islow can be characterized as a conductance mediating a late ca influx , but it can not unequivocally be grouped with soce . prolonged afterpotentials induced by chromatic adaptation have been described in some invertebrate photoreceptors ( prolonged depolarizing afterpotential ; hillman et al . , 1983 ) and attributed to the accumulation of metarhodopsin ( m ) in species where its spectral absorption differs substantially from that of rhodopsin ( r ) . however , prolonged depolarizing afterpotential is simply the prolongation of the photoresponse itself ( due to arrestin saturation ) , not a separate , gradually developing process like islow . moreover , in lima microvillar photoreceptors , the lack of aftereffects of intense chromatic stimulation suggests that there is no significant spectral shift in the transition rm , and hence no likely accumulation of m that may trigger the observed outcome by some other means . in conclusion , islow mediates a sustained influx of calcium that occurs under stimulation conditions that lead to calcium store depletion , and may thus serve the purpose of sustaining calcium signaling and/or aiding in the refilling of the intracellular stores . however , its regulation seems largely independent of the luminal levels of er calcium . the large size and reproducibility of islow make it a useful model system to investigate the control mechanism of receptor - operated currents , which at present remain poorly understood .
introductionfood security has been defined as the availability , stability , access and utilization of safe foods . diabetes has been known as one of the biggest health and medical problems throughout the world and is clearly related to lifestyle , and particularly , improper food consumption . the aim of this study was to determine the relationship between food security with sugar and blood pressure in patients suffering from type 2 diabetes who refer to diabetes centers in tehran.methodsthis cross - sectional study was conducted in 2015 on type 2 diabetes patients in tehran , iran . from two diabetes centers in the eastern and southern parts of tehran , 243 type 2 diabetes patients were selected . necessary information ( demographic and food security information ) about all the studied persons was collected using the standard questionnaire verified by us department of agriculture ( usda ) . the data was analyzed by spss version 16 , statistical comparisons were made using analysis of variance ( anova ) and chi - square and tukey tests and a significant level of < 0.05.resultsmost subjects were female ( 68.7% ) . there was no significant relationship between gender and food security ( p=0.372 ) . no significant relation was observed between food security and fasting blood pressure , hba1c , and systolic blood pressure ( p>0.05 ) , but there was a significant relationship between food security and diastolic blood pressure ( p= 0.030).conclusionsaccording to the relationship between diastolic blood pressure and food security and the role of blood pressure in the irreparable diabetic complications , it is recommended to perform appropriate food advice .	food and nutrition are basic human needs and providing them is directly related to food security . food security refers to peoples access to enough food at all times , in order to have a healthy and active life , which includes availability of enough healthy and nutritious food , and capability and assurance of obtaining suitable foods via acceptable ways ( 1 , 2 ) . studies of global organizations also indicate that more than 800 million people in developing countries do not have enough food and 34 million people in developed countries suffer from food insecurity ( 3 ) . a study in iran also explains the concern that 20% of people in society do not have the necessary financial capability for feeding themselves and about 50% have problems in terms of supplying their cell nutrition . in other words , one fifth of people suffer from lack of energy and half of them suffer from lack of micronutrients ( 4 ) . researchers believe that people suffering from food insecurity are exposed to its negative effects on their health level ( 5 ) . different studies have also reported a higher prevalence of some risk factors of non - communicable diseases such as overweightness , obesity , diabetes , hypertension ( 6 ) , and smoking ( 7 ) in the populations with low food security . type 2 diabetes is a kind of common metabolic disorder which is characterized by having disorders in glucose homeostasis and the insulin discharge and operation . in addition to decreasing life quality and imposing medical expenses , type 2 diabetes , which constitutes more than 90% of cases suffering from diabetes , increases mortality by 24 times ( 8) . the prevalence of this disease is increasing in both developed and developing countries ( 9 ) . more than 230 million people around the world suffer from diabetes and the highest spread of this disease is in the eastern mediterranean and middle eastern regions with 592 million people ( 10 ) . iran , as one of the countries in the middle east , has 3.5 million diabetic patients ( 11 ) . diabetes is clearly related to lifestyle , especially unsuitable food consumption and low body activity ( 12 , 13 ) . by reason of a most extensive spread of food insecurity ( 14 , 15 ) and growing spread of type 2 diabetes in iranian society ( 16 ) and the direct relationship between this disease and the quality and quantity of food diet ( food security ) together with the point that , in iran , there have been few studies on food security among diabetic patients , this study was conducted to determine the relationship between food security with blood sugar and blood pressure among diabetic patients . this cross - sectional study was conducted in eastern and southern diabetes centers of tehran , capital city of iran in 2015 . to determine the sample size , initially , based on this pilot study , the correlation coefficient and food security score were obtained as 0.18 . the first type error and test power the inclusion criteria were being older than 25 years old , consent for entering the study , and tendency for continuing the cooperation during the questioning process . in scoring the 18-item family food security questionnaire , a positive score ( 1 score ) , sometimes correct , almost every month , some months , and yes , and a zero score was given to the responses incorrect , the 18- item food security questionnaire of us department of agriculture ( usda ) ( 17 ) has been translated and validated in iran ( 15 , 18 ) . also , after 12 hours of fasting , 3 cc venous blood samples were taken from each patient using a 3cc syringe . all the samples were gathered during 79 a.m. and analyzed in the laboratory of the diabetes center . blood pressure ( bp ) was measured on the left arm , in the sitting position , using appropriate sized cuffs by using a calibrated electronic bp device ( welch - allyn ) . circumferences were measured using a plastic tape measure at the following levels : smallest waist circumference , umbilicus level , widest hip circumference , hips at the level of the anterior superior iliac spine of the iliac crest , and the highest thigh circumference . data were collected through questionnaires , biochemical and anthropometric ways to determine the relationship between food security with sugar level and blood pressure in diabetes type 2 . statistical analyses of the data were done with spss software version 16 ( spss inc . , the data were statistically analyzed with chi - square , one - way analysis of variance ( anova ) and post - hoc analysis of variance . informed consent had been obtained from all subjects before the study and patients names were omitted . this cross - sectional study was conducted in eastern and southern diabetes centers of tehran , capital city of iran in 2015 . to determine the sample size , initially , based on this pilot study , the correlation coefficient and food security score were obtained as 0.18 . the first type error and test power the inclusion criteria were being older than 25 years old , consent for entering the study , and tendency for continuing the cooperation during the questioning process . in scoring the 18-item family food security questionnaire , a positive score ( 1 score ) , sometimes correct , almost every month , some months , and yes , and a zero score was given to the responses incorrect , the 18- item food security questionnaire of us department of agriculture ( usda ) ( 17 ) has been translated and validated in iran ( 15 , 18 ) . also , after 12 hours of fasting , 3 cc all the samples were gathered during 79 a.m. and analyzed in the laboratory of the diabetes center . blood pressure ( bp ) was measured on the left arm , in the sitting position , using appropriate sized cuffs by using a calibrated electronic bp device ( welch - allyn ) . circumferences were measured using a plastic tape measure at the following levels : smallest waist circumference , umbilicus level , widest hip circumference , hips at the level of the anterior superior iliac spine of the iliac crest , and the highest thigh circumference . data were collected through questionnaires , biochemical and anthropometric ways to determine the relationship between food security with sugar level and blood pressure in diabetes type 2 . statistical analyses of the data were done with spss software version 16 ( spss inc . , the data were statistically analyzed with chi - square , one - way analysis of variance ( anova ) and post - hoc analysis of variance . informed consent had been obtained from all subjects before the study and patients names were omitted . out of 243 studied diabetic patients , 167 ( 68.7% ) were female and 76 ( 31.3% ) were male with an average age of 59 + 11.2 years old . in the present study , out of 167 women , 50 ( 29.9% ) were in the food secure group , 100 ( 59.9% ) were in the food insecure group without hunger , and 67 ( 10.2% ) were classified in the food insecure group with hunger . also , out of 76 studied men , 27 ( 35.5% ) were in the food secure group , 45 ( 59.2% ) were in the food insecure group without hunger , and 4 ( 5.3% ) were categorized in the food insecure group with hunger . chi - square statistical analysis showed no significant relationship between gender and food security ( p=0.372 ) . although waist and hip circumference , diastolic and systolic blood pressures , and glycosylated hemoglobin were higher in the insecure food group than the secure food group , analysis of variance on age , waist circumference , hip circumference , average number of family members , duration of suffering from disease , blood sugar , and blood pressure showed a significant relationship between food security score and diastolic blood pressure ( p=0.03 ) ( table 1 ) . in table 2 , tukey s post - hoc analysis of variance is done which shows a significant difference in terms of systolic blood pressure between the food secure group and food insecure group without hunger . in this study , waist and hip circumference , diastolic and systolic blood pressures , and glycosylated hemoglobin in the food insecure group were more than those of the food secure group , but this difference was significant only in terms of diastolic blood pressure . a part of the relationship between food insecurity and non - communicable diseases is probably due to the effects of food insecurity on food patterns , because it leads the suffering population toward consumption of cheap foods which have high density and low value in terms of micronutrients ( 19 ) and having a food pattern with low health level . this pattern can lead to the appearance of metabolic diseases such as diabetes ( 14 ) . a study by gucciardi et al . showed that food insecurity had 9.3% higher prevalence among diabetic patients than those not suffering from diabetes ( 6.8% ) ( 20 ) . in another study , the prevalence of diabetes in the food secure , slightly insecure , and extremely insecure groups was respectively reported as 11.7% , 10% , and 16.1% , which showed that its prevalence was higher among the people with extreme food insecurity ( 21 ) . in 2012 , a study was performed on 711 diabetic patients , among whom the spread of food insecurity was 46% . furthermore , among these people , weaker hba1c and blood sugar control were reported ( 22 ) . in the study done by david et al . , the relationship between systolic blood pressure , tc , fasting blood sugar , hba1c , and food insecurity was investigated . this study showed that the above cases had no relationship with food insecurity , but body mass index ( bmi ) was related . it also represented that 48% of food insecure people were obese , but only 35% of people with food security were obese . the study revealed that changing the nutrition pattern could probably cause obesity in people with food insecurity ( 6 ) . therefore , the results of holben et al.s study did not verify the results of the present work . in the present study , the blood pressure of diabetic persons was related to food security , but food security had no significant relationship with the body mass index ( bmi ) of the patients , which demonstrated that increased pressure in such patients was not because of their weight gain . in the cross - sectional study carried out on 135 diabetic patients in shiraz , the spread of food insecurity in the studied population was 66.7% . in this study , food insecurity showed a significantly direct relationship between high bmi , overweight , and obesity , but the variables of family background of disease , height , age , and gender had no relationship with food insecurity ( 18 ) . similar to the present study , it showed that the spread of food insecurity among the diabetic patients was high ; but in the present study , it was shown that food security had no relationship with bmi . all over the world , studies related to food security have been typically related to obesity . a study has shown that food insecurity has a significantly positive relationship with obesity ( 23 ) . whereas another work has demonstrated that food and weight insecurity have no correlation ( 18 ) . in the study in malaysia , carried out on the women of families suffering from food insecurity in rural regions , a probable relationship was found between waist circumference and food security ; which was not true about obesity ( 24 ) . in the study by morales et al . about food security among mexican women , the relationship between insecurity and obesity was studied and obesity in women with food insecurity was found to be higher than that among the secure women ( 25 ) . in contrast , a study done in columbia showed that food insecurity could be only a predictor of low weight in adults and it had no relationship with overweightness ( 26 ) . in sum , the results showed a significant relationship between food insecurity and hypertension in the diabetic patients . according to the high spread of food insecurity among diabetic persons , food insecurity must be considered one of the effective factors for the food pattern and prevention of irreparable diabetes complications . also , it is recommended to present suitable food advice for the prevention from diabetes complications .
backgroundmetagem is a wide gender - medicine project comprising post hoc and meta - analyses by gender of clinical outcomes , therapeutic approaches , and safety data from previously conducted observational studies to explore possible gender differences in real - life clinical settings . we report the results of the safety meta - analysis of seven metagem studies , evaluating gender differences in adverse event ( ae ) incidence and severity.methodsdata were collected between february 2002 and july 2013 . male and female patients were compared for the main safety variables , using student s t - test , 2 test , or fisher s exact test as appropriate . as supportive analysis , a logistic regression model was estimated to evaluate associations between gender and outcome.resultsin total , 4,870 patients ( 46% females , 54% males ) were included in the analysis ; age was higher for females ( mean standard deviation 61.218.3 years ) than males ( 56.316.6 years ) . overall , 264 aes were reported ( 59.1% in males ) . there were no significant gender differences in the percentage of patients with at least one ae : 3.0% for females versus 3.9% for males , 2 test p>0.05 . according to the logistic regression model results , no association between gender and aes occurrence seems to exist . a statistically significant gender difference in the percentage of drug - related aes emerged ( 37.6% in females vs 20.8% in males , 2 p=0.0039 ) . slightly significantly more aes in females were addressed with treatment compared with males ( 78.1% vs 66.7% , 2 p=0.0485 ) . total serious aes ( saes ) were 47 ( 72% in males ) . the frequency of patients with 1 sae was 0.6% in females versus 1.2% in males ( 2 test p=0.0246).conclusionthis safety analysis on a large sample of almost 5,000 patients with different diseases and treated with a wide range of different drugs provides a useful overview on possible gender differences in drug tolerability , which may be helpful in more accurately designing future clinical trials from a gender - specific perspective .	clinical data suggest that male and female patients exhibit differences regarding the pharmacology and toxicity of medications1 and differ in their response to drug treatment , not only as a result of physiological differences , such as body weight , surface area , and extracellular and intracellular water , but also in terms of differences in pharmacokinetics ( pk ) and pharmacodynamics ( pd).24 it is known that sex hormones influence drug absorption , distribution , metabolism , pd , and adverse events ( aes).5 in 2001 , on the basis of its adverse events reporting system , the us food and drug administration stated that females experience more and more serious aes ( saes ) than males.6 it has been hypothesized that this may be due to overdosing , different pk and pd , the fact that females are more likely to report aes than males , or that females take more medications than males . it has been shown that females use more medicines than males do and have a higher rate of chronic diseases , but they also generally pay more attention to their health and have more consciousness and care about themselves.6 despite the increasing evidence of physiological and pathological differences between genders , beyond those related to reproduction,711 females still represent a small percentage ( 22% ) of participants in phase i trials which are essential to verify drug dosage , aes , and safety.12 medical research has been conducted for decades with a large prevalence of male participants in clinical studies , yet the findings of these studies have often been applied to both genders.12 only with the new millennium has this changed with the european union promoting females s participation in research projects and the world health organization including gender medicine in the equity act in order to achieve gender - appropriate care.13 however , to date , the analyses provided by the pharmaceutical industry to the regulatory authorities often do not classify safety and efficacy data by gender . in 2013 , the italian drug agency ( agenzia italiana del farmaco ) invited pharmaceutical companies to process data divided by gender during the submission of regulatory documentation , so as to highlight possible differences.14 in the same year , novartis italy put in place a wide gender - medicine project , called metagem , which included analysis by gender of the data from nine previously conducted observational studies . these studies were performed between 2002 and 2013 , and covered a range of different clinical areas , including immune - mediated disorders ( psoriasis and psoriatic arthritis ) , transplantation medicine ( liver and kidney transplants ) , infectious diseases ( hepatitis b ) , and the central nervous system ( parkinson s disease and alzheimer s disease [ ad ] ) . the aim of the metagem project was to analyze and describe clinical outcomes , therapeutic approaches , and safety data by gender , using post hoc analyses and meta - analyses , in order to explore possible gender differences . the methodology of the metagem project has been described in detail elsewhere.15 the present paper reports the results of the overall safety analysis of the metagem studies , aimed at evaluating possible gender differences in the incidence and severity of aes and the potential association between gender and safety . seven of the nine observational studies of the metagem project were included in this safety analysis on 2,612 ( 53.6% ) males and 2,258 ( 46.4% ) females : they are listed and briefly described in table 1 . with regard to the two excluded studies , the gender - attention ( the female in her real dimension : effect of gender and hormonal status on adverse events incidence in psoriatic patients treated with cyclosporine ) study ( colombo et al , unpublished data , 2013a ; colombo et al , unpublished data , 2013b ; colombo et al , unpublished data , 2014a ; colombo et al , unpublished data , 2014b ; colombo et al , unpublished data , 2016 ) was not included because a gender - specific safety assessment was the primary objective of the study itself , while the studio osservazionale italiano per la valutazione dellinsufficienza renale in pazienti con trapianto di fegato ( surf ) ( italian observational study for the evaluation of renal insufficiency in liver transplant patients ) study ( donato et al , unpublished data , 2013 ) was also not considered because it did not include safety data collection . an ae was defined as any unfavorable or unintended sign , symptom , or disease that occurred from the time the signed informed consent was obtained until the end of the patient observation period . an ae was defined as a sae if it resulted in death , was judged life - threatening , required inpatient hospitalization or prolongation of existing hospitalization , resulted in persistent or significant disability / incapacity , or caused a congenital anomaly / birth defect in the child of the observed patient . in cases where such information was available , data were collected between february 2002 ( first patient first visit ; psychae study16,17 ) and july 2013 ( last patient last visit ; iceberg study [ rizzetto et al , unpublished data , 2011 ; bandiera et al , unpublished data , 2012 ] ) ; first patient first visit and last patient last visit for each study are shown in table 1 . monitoring visits were conducted to verify whether enrollment was performed consecutively and according to inclusion / exclusion criteria . moreover , before statistical analysis , a data cleaning process was run to check the collected data for completeness and accuracy . patients were considered evaluable for this analysis if they were evaluable for the individual study in which they participated and if gender was recorded in the case report form ( crf ) , as detailed elsewhere.1623 evaluable patients were described according to sociodemographic ( such as age ) and clinical ( such as ongoing specific therapies ) features . moreover , male and female patients were compared with regard to the main safety variables : ae occurrence and characteristics ( description , intensity , possible correlation with a drug , evolution , and therapeutic intervention , if any ) , and sae occurrence and description . comparisons were performed by student s t - test , test , or fisher s exact test as appropriate . for the post hoc analyses , all p - values presented are exploratory . patients with missing data in selected parameters were not excluded from analysis , but were simply not evaluated for these parameters . data collection was performed for all studies through the crf ; however , not all variables of interest were present in all studies or , if present , not all had the same answer categories . for these reasons , ae crf fields that were common to all studies were identified through a preliminary analysis , and a new variable for each outcome of interest ( eg , ae description , ae intensity ) was created considering the value in each study . different answer modalities were present in the original crfs for ae intensity , possible correlation of ae with a drug , and ae evolution , and so recoding was performed . briefly , the ae intensity was classified as mild , moderate , high , or not determined , as originally reported in most studies . in the evolution study , grade 1 intensity ( based on common terminology criteria for adverse events v3.0 ) was considered as mild , grade 2 as moderate , and grades 35 as high , while in the iceberg study ( rizzetto et al , unpublished data , 2011 ; bandiera et al , unpublished data , 2012 ) , aes defined as severe were considered to be of high intensity . for the cetra study , the intensity of aes could not be analyzed because this variable was not collected on the crf . possible correlation with a drug was defined as present ( yes ) , absent ( no ) , or not determined ; if changes in therapy had been introduced , these were evaluated in terms of dosage escalation or reduction , therapy discontinuation , or other . concerning their evolution , aes were considered as resolved ( including resolved without sequelae ) , unresolved , toward resolution , resolved without sequelae , or resolution unknown . saes were reported as death , hospitalization , disability , persistent or significant inability , life - threatening events , or not determined . as supportive analysis , a logistic regression model was estimated to evaluate the association between gender ( male , female ) and outcome ( the patient had at least one ae during the study ) . the model provided estimates of the odds ratios of experiencing an ae in female versus male patients . cary , nc , usa ; enterprise guide v4.3 copyright 20062010 by sas institute inc . , cary , nc , usa ) . in total , 4,870 patients , 46% females and 54% males , were included in the analysis , as detailed in table 1 . age by gender and study is reported in table 2 : overall , mean age was higher in females ( 61.218.3 years ) than in males ( 56.316.6 years ) . disease - specific therapies ongoing at enrollment in the studies are summarized in table 3 . overall , 264 aes were reported , 59.1% in males ; table 4 summarizes the type of aes , according to the medical dictionary for regulatory activities ( meddra ) system organ class . there was no significant gender difference in the percentage of patients with at least one ae ( ie , patients with 1 , 2 , or 3 aes ) : 3.0% for females versus 3.9% for males , test p0.05 . details about the occurrence and intensity of aes by gender and study are reported in table 5 . the results of the logistic regression model also showed no association between gender and ae occurrence : odds ratio = 0.764 ( females vs males ) , 95% confidence interval : 0.5591.044 , but a statistically significant gender difference in the percentage of drug - related aes emerged ( 37.6% in females vs 20.8% in males , test p=0.0039 ) . overall , more aes were addressed with some kind of treatment in females compared with males ( 78.1% vs 66.7% , the frequency of patients with 1 sae was 0.6% in females versus 1.2% in males ( test p=0.0246 ) . gender - specific medicine is the study of how diseases differ between males and females in terms of prevention , clinical manifestations , therapeutic approach , outcomes and tolerability , prognosis , and psychological and social impact . there has been increasing evidence in recent years of physiological and pathological differences between the genders . focusing on the diseases examined in the studies included in our analysis , many gender differences have been reported for patients with psoriasis and psoriatic arthritis , in terms of epidemiology,24 pattern and burden of disease,25 search for care,26 and choice of therapy;27,28 for example , peripheral joint involvement , pain , and functional impairment are more frequent for female than for male patients with psoriasis.2932 moreover , the psychae study16 pointed out that the psychological status of females was also worse than that of males , independently of the severity of psoriasis . there is also an extensive literature on gender differences in ad , as recently reviewed by li and singh.33 clinical studies have shown differences between males and females in specific cognitive ability domains and risk of ad at later age . several major biological hypotheses have been postulated , such as differences in age - related sex hormone reduction ( estrogens , progesterone , testosterone ) , impact from risks of other diseases ( diabetes , depression ) , and age - related decline in brain volume . in parkinson s disease as well , gender - related differences have been recognized , although these are still poorly understood . prevalence and incidence of parkinson s disease are significantly higher in males than in females.34,35 from a clinical point of view , females with parkinson s disease have shown worse capacity in activities of daily living and more severity of levodopa - induced dyskinesia in several studies,3638 while male gender was shown to predict worse rigidity score and higher risk for sleep behavior disorder , dementia , and death.37,3941 a 2014 cross - sectional survey42 found that males reported a greater disease burden and greater daily levodopa equivalent doses than females . the greater burden of disease score in males was significantly associated with gender even after controlling for age and disease duration . concerning transplants , gender - based disparities have been observed in post - liver transplantation outcomes , together with a continuous decline in the number of liver transplantations in females.43 the effect of gender on drug tolerability has begun to raise interest only in recent years , mainly resulting in the observation that females experience more frequent and more saes than males , possibly due to different pk and pd , higher rates of ae reporting , or greater use of medications in females compared to males.6,4446 clinical research sponsored by novartis italy in the past decade included several large observational studies in important medical areas , including psoriasis.16,17,22,47 central nervous system disorders , such as parkinson s disease and ad,1821 and transplantation,23 but none of those studies , except one ( the aforementioned gender - attention study [ colombo et al , unpublished data , 2013a ; colombo et al , unpublished data , 2013b ; colombo et al , unpublished data , 2014a ; colombo et al , unpublished data , 2014b ; colombo et al , unpublished data , 2016 ] ) , adopted a gender - specific approach in data analysis . based on the increasing interest in gender medicine and prompted by the large quantity of clinical data available through these large national studies , it was decided to reanalyze these data from a gender perspective ( the metagem project ) . the overall metagem sample ( n=4,870 ) consisted of 2,612 ( 53.6% ) males , with females being predominant only in the two ad studies , evolution and axept ( 59.7% and 63.6% , respectively , of those enrolled are female ) . this prevalence of female patients in the ad studies probably accounts for the overall higher mean age in females compared with males . there were no significant gender differences in the frequency of patients with at least one ae ( 3.0% for females vs 3.9% for males , test p>0.05 ) , with 59.1% of global aes occurring in males . considering only those reported as drug - related aes , the incidence was significantly higher in females , and this is consistent with several reports revealing that females are more prone to adverse drug reactions ( adrs ) than males , as confirmed by the evidence that eight of ten drugs that have been dropped out from the us market were responsible for more adrs in females than in males . indeed , risk factors for adrs , such as polytherapy , aging ( females become generally older than males ) , and depression , are in general more frequent in females than in males.4446,48,49 other factors hypothesized to explain why females usually report more adrs are the fact that females have a higher prevalence of pain ( headache , migraine , musculoskeletal pain ) and pay greater attention to their health status . moreover , physiological aspects , such as menstrual cycles , pregnancy , and menopause , are likely to have a relevant impact on the pk and pd of drugs . unexpectedly , we found that 2% of saes occurred in males , and the frequency of patients reporting at least one sae was significantly higher in males , despite the older mean age of females compared with males . these results are in contrast with the large quantity of published data showing that females experience more aes and that these are generally also more serious.6,3032 however , it has to be considered that the overall patient population was fairly unbalanced by the cetra study on renal transplantation ( inclusion criteria : age 18 years , renal allograft functioning for at least 6 months , and serum creatinine level < 2.5 mg / dl ) , which included 64% male patients and accounted for 77% of total saes ( 36 of 47 ) . this may be reasonably explained by the fact that the cetra patients were transplanted patients , frequently hospitalized for transplant - related events . for these reasons , in order to have a more homogeneous population , we repeated the analysis excluding cetra patients . this analysis showed no significant difference between the genders in terms of saes ( 0.2% of females vs 0.4% of males had at least one sae ; test , p>0.05 ) . we had no adequate information about the degree of disease severity in the patients considered for this analysis and therefore were unable to analyze for possible correlations between severity of disease and incidence of ae ; however , we hypothesized that the type and number of therapies administered could mirror the severity of the disease , at least for the deep study ( parkinson s disease ) and the psychae and synergy studies ( psoriasis psoriatic arthritis ) . following specific analyses , we found no correlations between the incidence of aes and the number of drugs administered to patients ( 12 and 3 ) in the deep study , or the type of therapy ( topical or systemic ) in the other two studies in psoriasis . this seems to confirm that the observed gender differences were not biased by the severity of the disease . it was not originally designed to assess gender differences in patients safety profile , and statistical analysis was mainly descriptive with only explorative p - values . moreover , owing to the inclusion of several studies , large differences in patient population , study design , and ongoing treatments have to be taken into account . for this reason , a common approach for data cleaning , recoding , and statistical analysis was followed . finally , one single study , cetra , which studied the quite complex and serious clinical condition of renal transplantation , accounted for the majority of aes ( 183/264 ) and saes ( 36/47 ) , and this may have strongly affected the results of our analysis . this safety analysis on a large sample of almost 5000 patients affected with different diseases and treated with a wide range of different drugs provides a useful overview , within the considered disease areas , of possible gender differences in drug tolerability , which may be helpful in more accurately designing future clinical trials from a gender - specific perspective .
aim : to assess the microtensile bond strength of repaired composite resin that was surface treated by diamond point or silicon carbide followed by bonding using either only total- etch bonding regimen or silane coupling agent with adhesive resin.materials and methods : fourteen composite blocks were aged under deionized water for 14 days . the bonding surface was prepared with coarse diamond point or silicon carbide . two blocks with no surface treatment were used as control groups . the bonding regimen was either total - etch bonding regimen or silane coupling agent and bonding agent . the aged samples were then bonded to new composite . five sections per block ( each 1 mm thick ) were prepared ; cut to obtain an adhesive zone of approximately 1mm2 and subjected to microtensile bond strength testing.results:the highest bond strength was obtained by surface treatment by coarse diamond point and total etch bonding regimen and least by silicon carbide and silane . a statistically significant difference was seen in all the four groups.conclusions:surface treatment by a coarse diamond point and total - etch bonding regimen provides highest bond strength . thus , a simpler treatment regimen can contribute to a better bond strength in repaired composites .	resin based dental composites are gaining a wide acceptance in today 's dental practice . in clinical practice the unreacted c = c bonds of the functional groups on the surface of a polymerized matrix will allow the monomer of new resin composite to bond , thereby improving adhesion . but , over time these materials undergo degradation and deterioration in the intra - oral environment . these processes lead to fracture or defects of the composite restoration either at the composite - tooth interface or within the restorative material itself . for years , the traditional management consisted of replacing entire restorations . more recently a minimally invasive operative philosophy that aims to repair such defects ; which is more conservative , less traumatic to pulp , less time consuming and more cost - efficient or at least can be used as an acceptable interim procedure until further treatment has become a viable option . due to of limited number of reactive methacrylate groups after polymerization and water sorption into pre - existing composite , the repaired composite can not effectively bond to the aged composite restoration without adequate surface treatment . in such cases , providing sufficient bonding to the old restoration is important , and can be achieved either mechanically or chemically . though studies have been undertaken to find out optimal repair protocols using different bonding techniques ; there exists a wide gap between experimental methods and those used in daily restorative practice . hence , in the present study surface treatment by diamond point and silicon carbide , which are most widely used for finishing composites are compared along with total etch bonding regimen and silane coupling agent with bonding agent . fourteen composite blocks of a microhybrid composite resin ( esthet x hd ; dentsply india ) { shade b2 } were made ; each measuring 8 mm 8 mm 6 mm using teflon molds . from each block ; 5 samples was prepared . it was then categorized into 1 control ( 2 blocks ) and 2 experimental groups ( 6 blocks for each group ) . the grouping of samples is as follows : group a ( control group ) ( n=10)-no surface treatment subgroup a1 ( n=5)- the bonding surface was etched ( frost ; ammdent , india ) and bonding agent ( adper single bond ; 3 m , espe , usa ) applied . subgroup a2 ( n=5)- bonding surface was treated with silane primer ( rely x ceramic primer ; 3 m , espe , usa ) and bonding agent applied . group b ( experimental group ) ( n=30)-coarse diamond point { ( 125 m to 150 m ) ; mani inc , japan } was used for surface treatment . surface was abraded two times for 5 sec by the same operator to maintain standardization . group c ( experimental group ) ( n=30)- silicon carbide { ( green carborundum stone ) ; shofu dental corporation , japan } was used for surface treatment . surface was abraded two times for 5 sec by the same operator to maintain standardization . subgroup c2 ( n=15 ) - application of silane primer and bonding agent . after surface treatment the blocks were subjected to ultrasonic cleaning for 10 mins to remove surface debris and ensure proper adhesion of composite . following the surface treatments and bonding regimen applications , the aged composite blocks ( shade b2 ) an increment of 2 mm was obliquely layered and light cured using a halogen light - curing unit . ( translux energy , heraeus kulzerinc , germany ) for 20 sec per increment at 600 mw / cm . 5 sections per block each ( thickness of per section-1 mm ) were prepared by a slow speed diamond saw ( isomet saw ; buehler lake bluff , illinois , usa ) . hourglass shaped specimens with a cross - sectional area of 1 mm were made and subjected to microtensile bond strength in a universal tester ( instron 3365 ) . the results of the microtensile bond strength are summarized in table 1 . a comparison between various groups means , sd , minimum and maximum load for different subgroups t - test value for comparison between subgroup a1 and subgroup a2 t - test value for comparison between subgroup a1 and subgroup b1 t - test value for comparison between subgroup a1 and subgroup c1 test value for comparison between subgroup a2 and subgroup b2 t - test value for comparison between subgroup a2 and subgroup c2 t - test value for comparison between subgroup b1 and subgroup b2 t - test value for comparison between subgroup c1 and subgroup c2 comparison of control and experimental subgroups resin - based composites are widely employed in dentistry since the advent of adhesive technologies . adhesive dentistry brought into perspective the possibility of a more conservative approach for tooth restoration , based on the reduction of the cavity preparation size and the bonding of the restorative material ( resin - based composite ) to tooth structure . hence , a feasible treatment option of such failed restorations can be the repair of just that failed part ; rather than the replacement of the entire restoration . repair preserves the sound tooth structure that would be at risk during replacement of the restoration and also prevents repeated trauma to the pulp - dentin organ . however , repair of an existing restoration is also not bereft of all flaws . the substrate to be bonded is a chemically degraded one with no free radicals to bond to another layer of fresh composite . commercially the available bonding systems ; rely on primarily adhesion to either enamel or dentin with none , formulated specifically to bond to the non - mineralized aged resin - based composite substrate . bonding to this substrate depends on various surface roughening techniques such as with air abrasion , diamond points or acids and the use of intermediary agents such as adhesive resins and silane primer . the resin composite used in this study ( esthet x hd ) is a microhybrid composite with a particle size of 0.6 to 0.8 m and loading 77% ( by wt % ) or 60% ( by vol % ) . it belongs to one of the most commonly used resin composite family , and has been claimed by manufacturers to be used as an universal restorative material ( for both anterior and posterior restorations ) . various techniques have been utilized to simulate the intra - oral aging conditions.[121618 ] among all methods , aging due to immersion in water has generated maximum research and is most commonly seen in oral cavity . water diffusion through the polymer chains and boundaries with fillers and the hydrolytic deterioration of the polymer chains resulted in elution of components and the plasticization of the composite . initially , this process would affect surface properties , such as hardness and wear resistance . however , with time , it also interferes with the bulk properties , such as the strength and fracture toughness of the material thereby , compromising the long - term durability of the restoration.[2022 ] hence , in this study , aging by immersion in water for 14 days was used . it is rather easy to bond to fresh composite , which is covered by an oxygen inhibition layer , because a covalent bond is established between the uncured surface layer and the newly applied composite material . aged composite materials lack this zone , and hence other viable alternatives to facilitate bonding of the new materials with the aged composite needs to be investigated . in this study , three different surface treatments and bonding regimens were applied according to various alternatives;[2325 ] creating a micro - mechanical locking substrate using a coarse diamond point or silicon carbide . forming a potential interpenetrating network using a total - etch bonding regimen . creating chemical bond to the surface of exposed glass filler surfaces by application of silane to the surface . although , the effect of different contemporary chemical and mechanical treatment procedures on the repair bond strength of aged composite were evaluated previously , the choice of the different chemical and mechanical repairing procedures in this study were aimed to evaluate the validity of the simplest and most common methods that were used in every day clinical practice . defects in the old composite resin restorations may be restricted only to the old composite resin or may involve the composite resin and the adjacent tooth structure as well . when such defect involves both the restoration and the tooth , additional preparations in the form of removal of discolored or carious tooth tissues and/or bevel preparation is recommended . in the daily dental practice , two different abrasives have been used : coarse diamond point.silicon carbide . coarse diamond point . the micro - mechanical roughening produced by these abrasives depends on the abrasive particle size . this was done to remove any superficial debris produced by the mechanical roughening , which might hinder the bonding to aged composite resin . the highest repair bond strength obtained by the use of total - etch bonding systems can be attributed to : the unfilled resins in the bonding agent penetrated into the roughened composite surface , creating a new oxygen inhibited layer . the bonding agent formed covalent hydrogen bonds to the silane in the inorganic filler particles . in the present study , silane pretreatment has shown lower bond strength when compared to total - etch bonding regimen and can be attributed to : mechanism through which fillers react when mechanically loaded , aged or abraded is by breaking off in clusters . this loss of fillers might reduce interaction with silanes and thus decrease the bond strength . incomplete solvent evaporation during air - drying may occur resulting in increased viscosity of the adhesive blend exerting an adverse effect on the polymerization . because of surface treatment ; the silane layer on any remaining filler is depleted . thus , chemical adhesion of the unpolymerized resin matrix to these inorganic particles was unlikely . following the surface treatments and bonding regimen applications , the aged composite blocks ( shade b2 ) were bonded to new composite ( shade a3.5 ) . this made it easier to prepare an hour - glass shaped specimen by cutting along this adhesive zone or interface . considering the afore - mentioned facts , surface treatment by coarse diamond point followed by etching and bonding resin application produces the highest microtensile bond strength in comparison to either surface treatment by silicon carbide or application of silane primer . from a clinical point of view this might be advantageous , since the use of silane may interfere with composite bond to enamel and /or to dentin exposed at the repair site . however , in applying the findings of an in vitro study to a clinical situation , it is important to realize that the two situations are widely different and may not co - relate completely . i affirm that , i have no financial affiliation ( e.g. , employment , direct payment , stock holdings , retainers , consultantships , patent licensing arrangements or honoraria ) , or involvement with any commercial organization with direct financial interest in the subject or materials discussed in this manuscript , nor have any such arrangements existed in the past 3 years . any other potential conflict of interest is disclosed . within the limitations of this study it can be concluded that : bond strength obtained by surface treatment with coarse diamond point is significantly higher than that obtained by surface treatment with silicon carbide . total - etch bonding regimen produces higher bond strength compared to treatment with silane primer and bonding resin application . the highest bond strength is achieved by surface treatment with coarse diamond point followed by application of total - etch bonding regimen .
we report a systemic mycosis in a german shepherd dog caused by scytalidium spp . the patient presented for progressive cervical pain and forelimb hemiparesis . cervical computed tomography revealed lysis associated with multiple vertebrae and a soft tissue mass adjacent to the spinal cord , as well as prescapular lymphadenopathy . fine needle aspirates of the lymph nodes yielded hyphae , and a subsequent culture obtained a scytalidium spp . itraconazole therapy was initiated , but the subject was euthanized three months later due to progressive neurologic disease and discomfort . this appears to be the first report of disseminated disease by this species in veterinary medicine .	in veterinary medicine , numerous mycotic agents have been reported to cause systemic infections , often affecting the central nervous system [ 16 ] . german shepherd dogs appear to overrepresented in this subset of veterinary literature , and are reportedly predisposed to opportunistic fungal infections . in current literature , a case of systemic infection cause by scytalidium species has not been reported in veterinary medicine . in humans , several subspecies of scytalidium including scytalidium hyalinum and neoscytalidium dimidiatum , have been documented as responsible for human cases of dermatomycosis , infections that mimic dermatophytosis . scytalidium infections occur in the feet and nails of people walking barefooted in contaminated soil in tropical countries such as thailand , nigeria , australia , and jamaica . rare systemic infections have been reported involving central nervous system abscesses , endopthalmitis , sinusitis , osteomyelitis , mycetoma , subcutaneous lesions , and disseminated infections , usually in immunocompromised or immunosuppressed patients . these fungi show a high resistance to antifungal drugs used most commonly in human and veterinary medicine . we describe a case of disseminated mycosis leading to progressive cervical pain and forelimb hemiparesis in a german shepherd dog caused by scytalidium spp . a four - year - old intact female german shepherd dog was referred to the surgery center for a month long history of cervical pain and progressive forelimb hemiparesis . on initial physical exam ( day 0 ) , she was found to have mildly delayed proprioceptive placing in her right pelvic limb . she had been treated with steroids by the primary veterinarian , and was initially responsive , but had begun to exhibit mild neurologic dysfunction in her forelimbs ( stumbling and mild toe scuffing ) in the week prior to presentation . computed tomography ( ct ) of the patient 's cervical spine showed lysis of the endplate with active periosteal reaction of the ventral aspect of the fourth vertebral body ( c4 ) ( fig . similar lysis was noted in the end plates of the fifth and sixth vertebral bodies ( c5 and c6 ) . a soft tissue mass was noted along the left lateral aspect of the vertebral canal through the body of c5 ( fig . 2 ) . the soft tissue did not overlie an intervertebral disc space , but did cause dorsal and lateral deviation of the spinal cord . severe symmetrical bilateral pre - scapular lymphadenopathy and moderate bilateral retropharyngeal lymphadenopathy were also present ( fig.3 ) . following imaging , the initial primary differential diagnosis for the patient s condition was metastatic neoplasia , based on the combination of the soft tissue mass adjacent to the spinal cord and the generalized lymphadenopathy . secondary consideration was given to discospondylitis with associated reactive lymph nodes , though the presence of a soft tissue mass is not a typical finding in patients with discospondylitis . cerebral spinal fluid ( csf ) and aspirates from the pre - scapular lymph nodes were sent for cytologic evaluation . the patient was discharged the following day ( day + 1 ) with carprofen 2.2 mg / kg po twice daily ( bid ) and tramadol 3 mg / kg po three times daily ( tid ) for palliative care pending cytology results . the pre - scapular lymph node aspirates were indicative of systemic fungal infection , yielding moderate reactive lymphoid hyperplasia with mild to moderate pyogranulomatous inflammation containing intralesional fungal hyphae ( fig . 4 ) . based on cytology results , a second lymph node aspirate sample was submitted for fungal culture . itraconazole therapy was initiated at 5 mg / kg po bid , and gabapentin 5 mg / kg po tid was added for additional pain control ( day + 5 ) . the lymph node aspirate was plated on four different fungal culture media : sabouraud 's dextrose agar , potato dextrose agar , mycobiotic agar , and trypticase soy agar with 5% sheep blood . a pure culture of fine , filamentous fungal growth was observed on the sabouraud 's dextrose , potato dextrose , and blood agars , while no growth was observed on the mycobiotic agar . by the sixteenth day of incubation , the three agars on which the fungal isolate grew were covered with dark gray to black wooly growth . microscopic examination of a wet mount of the fungal hyphae revealed the formation of chains of arthroconidia . dna sequencing was performed on an approximately 475-base pair fragment of the intergenic transcribed spacer ( its ) region of the 18s ribosomal subunit . 5.0 l of extracted dna was amplified in a 25 l reaction volume with 20 l of pcr master mix containing its forward primer 5-tccgta ggt gaa cct gcg g-3 and its reverse primer 5-tcc tcc gct tat tga tat gc-3. reactions were performed on a 9700 thermocycler geneamp pcr system ( applied biosystems , carlsbad , california 92008 , usa ) . cycle sequencing on purified products was performed on a 3130 genetic analyzer ( applied biosystems ) . sequence data analysis was performed with the use of geneious pro software ( biomatters ltd . a blast search of the its sequence data in genebank at the ncbi website ( http://www.ncbi.nlm.nih.gov/blast/ ) revealed 95% identical sites with scytalidium lignicola ; the next closest match was 88.4% identity with scytalidium cuboideum . the morphologic evaluation combined with the molecular analysis led to the conclusion that an undetermined species of scytalidium had been isolated from the lymph node aspirate . three weeks after starting itraconazole and gabapentin therapy ( day + 21 ) , the owner reported that the patient was comfortable , and seemed improved neurologically . regrettably , three months ( approximately day + 60 ) after initiating antifungal therapy , the patient was euthanized due to declining neurologic status and comfort . there are more than 15 species of scytalidium listed , though only two are significantly associated with human pathology the pathogen generally causes superficial infections similar to dermatophytosis in the nail beds and skin of people living in tropical regions ( africa , south america , the caribbean , and parts of asia ) who come in contact with contaminated soil . recently , more cases of scytalidiosis have occurred in humans living in temperate countries , likely secondary to increased travel and immigration . prevalence in endemic areas ranges from 9 to 24% . deep or systemic infections are rarely reported in human literature , but can occur in immunocompromised patients . manifestations of systemic disease include central nervous system abscesses , endophthalmitis , sinusitis , and osteomyelitis . diagnosis is achieved via fungal culture of the affected area . according to machouart et al . ( 2013 ) , there is not a standardized therapy for treatment of scytalidium spp . since the organism is resistant to most topical or systemic antifungal agents used in human dermatology . in the case presented , the patient had no known travel history to an endemic , or tropical , region . she was not considered immunocompromised prior to diagnosis , though she was a breed considered predisposed to opportunistic fungal infection . her clinical signs and diagnostic imaging results were consistent with other case reports of dogs with vertebral osteomyelitis , spinal cord compression , and lymphadenopathy secondary to systemic fungal infection [ 16 ] . unsurprisingly , considering the reported resistance of scytalidium to antifungals , itraconazole therapy was not successful . in conclusion , the case report describes scytalidium spp infection causing vertebral lysis and spinal cord deviation leading to progressive cervical pain and forelimb hemiparesis . treatment was not successful ; leading to the suggestion that further diagnostics involving antifungal therapy targeted at scytalidium is warranted . to the author 's knowledge , this is the first report of systemic mycotic infection caused by scytalidium spp . in veterinary medicine .
abstractlots of athletic skills performed during practice or competition are initiated by the legs , where athletes either walk or run prior to executing specific skills . kinematic chains are used to describe the relationships between body segments and joints during movement . the aim of this study was to determine the relationships between movements of lower limb segments and the pelvis in open and closed kinematic chains while walking . the experimental group consisted of 32 males ( age 23.3 2.5 years , body mass 78.1 8.7 kg , body height 182 6 cm ) . for 3d analysis , an optoelectronic system vicon mx ( 7 cameras , frequency 200 hz ) was used . positioning of the segments was determined by the plugingait model . each participant executed five trials at speeds ranging from 1.38 to 1.52 ms-1 . the relationships between angle variables of the lower limbs and the pelvis in selected gait cycle phases were evaluated using statistica software ( version 10.0 ) and the spearman correlation . the highest numbers of moderate and large correlations were found at opposite toe off , heel rise and initial contact for the sagittal and transversal planes in comparison to the frontal plane . the closed kinematic chain had a stronger impact on determining the movement pattern . the instructions or interventions focusing on closed kinematic chain alternation are more effective for changes in a movement pattern . the preferred limb initiates kinematics in the direction of propulsion , while the non - preferred limb in internal and external rotation .	athletes either walk or run prior to executing specific skills ( panariello , 1991 ) . the gait is a complex movement which includes both the open and closed kinematic chain . therefore , the gait can be influenced by many factors such as age , the activity level , muscle activity ( bizovska et al . stastny et al . , 2014 , 2015 ) , injury ( bunton et al . , 1993 ) and disease ( krawczyk et al . , 2012 ; marchewka and chwala 2007 ) . in many studies , kinematic ( franz et al . , 2009 ) and kinetic ( bovi et al . , 2011 ; cigali et al . , 2004 ; jandaka et al . , 2013b ) variables during a gait cycle have been observed , but they were usually assessed independently in each joint . from a mechanical point of view , while walking a human body can be considered a system of segments and joints forming various types of chains depending on the phase of a gait cycle . the first works on kinematic ( kinetic ) chains in this field were published in the 1870s . the authors of that time described kinematic chains as a system of overlapping rigid bodies linked by pivots . upon external loading , force was transferred from one segment to an adjacent segment , thereby enabling cumulative response of the whole chain ( reuleaux , 1875 ) . in the 1950s , arthur steindler observed the possibilities of applying kinematic chains in the motor system ( steindler , 1977 ) . one of the reasons for his interest was the finding that muscle activity in the lower limb with the foot fixed to the ground ( a closed chain ) was different when the foot was in free motion ( an open chain ) , what also has implications in sports training . in an open kinematic chain ( okc ) , the joint angle can be adjusted without incurring any changes in other joints . in a closed kinematic chain ( ckc ) , however , both ends of the chain are fixed and any adjustment in the angle in one joint reciprocally results in altered angles in the other joint . a human gait , as other complex movements , typically consists of both the open and closed kinematic chains that frequently alternate during movement of the lower limbs . in the swing phase , the limbs adopt an open kinematic chain such that the distal segment movement velocity relates to the velocity of the proximal segment . additionally , inertia acceleration in the distal segments is associated with deceleration of the proximal segments ( mcmullen and uhl , 2000 ) . during the stance phase ( ckc ) , stability of the proximal segments depends on the stability afforded by the distal segments and foot instability influences the ability to stabilize the trunk along with proprioception from fixed parts of the body ( bunton et al . , 1993 ) . in the scientific literature we can find many studies which observe interdependence between the movements of different limb segments during various movement tasks both in sport ( jandaka et al . , 2013a ; sasaki et al . , 2015 ) and clinical cases ( bunton et al . , 1993 ; khamis et al . , 2015 ; wright et al . , 2008 ) . however , when focused on walking , the authors mainly observed the relationships between parts of the foot and shank or knee ( dubbeldam et al . , 2013 ; pohl et al . , 2007 ) . our study is more comprehensive and includes movements at the ankle , knee , hip and pelvis . with regard to sports training , a crucial question arises whether the ckc or okc alternation would lead to parallel changes in the whole movement pattern . the aims of the study were to describe the relationships between movements of the lower limb segments and the pelvis in open and closed kinematic chains while walking and to assess the effects of laterality on these relationships . the experimental group consisted of 32 males ( age 23.3 2.5 years , body mass 78.1 8.7 kg , body height 182 6 cm ) . only participants with good health and no history of serious injury the preferred limb was determined prior to measurements by asking which leg the participant would use to kick a ball . for 3d analysis , a vicon mx ( vicon motion systems ltd . , oxford , uk ) optoelectronic system with seven infrared cameras ( type t10 , resolution 1 megapixel , frequency 200 hz ) was used . the participants were asked to walk on an 8 m long walkway including two force plates ( type : 9286aa , kistler instrumente ag , winterthur , switzerland ) . the positions of the lower limb segments and the pelvis were determined using the plugingait model ( 16 markers ) with consideration of marker placement error ( szczerbik and kalinowska , 2011 ) . this velocity range was determined during a preliminary study as the natural speed of the walk in young male subjects . angles at the ankle , knee , hip and pelvis in the sagittal , frontal and transversal planes were evaluated at specific gait cycle phases , as determined by force plates and the kinematic system as follows : initial contact ( ic),opposite toe off ( oto),heel rise ( hr),opposite initial contact ( oic),toe off ( to),maximal knee flexion ( mkf),tibia in the vertical position ( tv ) . initial contact ( ic ) , opposite toe off ( oto ) , opposite initial contact ( oic ) , maximal knee flexion ( mkf ) , tibia in the vertical position ( tv ) . ankle movement in the frontal plane ( inversion / eversion ) was not assessed since applying the conventional gait model in the absence of any enhancement in the set of foot markers is not considered reliable for this variable . the kinematic data were processed using standard procedures by vicon nexus and vicon polygon software ( vicon motion systems ltd . , the relationships between angle variables of the lower limbs and the pelvis ( table 1 ) in specific gait cycle phases were determined using statistica software ( version 10.0 , statsoft , inc . the kolmogorov - smirnov test showed a non - normal data distribution , thus , the spearman correlation coefficient was used . a correlation was considered large for values greater than 0.5 and moderate for values between 0.3 and 0.5 ( cohen , 1988 ) . positive correlations meant that movement in the joints occurred in the same direction ( both for positive or negative ) . for example , a positive correlation between the ankle and the knee in the sagittal plane indicated that increased ankle dorsal flexion was associated with increased knee flexion . conversely , a negative correlation illustrated a contrary joint movement ; for example , in the transversal plane there was internal rotation at the ankle and external rotation at the knee . description of measured angle variables positive positive angle values , negative negative angle values for clarity , our study focused only on adjacent segments and the individual assessment of each plane . the experimental group consisted of 32 males ( age 23.3 2.5 years , body mass 78.1 8.7 kg , body height 182 6 cm ) . only participants with good health and no history of serious injury the preferred limb was determined prior to measurements by asking which leg the participant would use to kick a ball . for 3d analysis , a vicon mx ( vicon motion systems ltd . , oxford , uk ) optoelectronic system with seven infrared cameras ( type t10 , resolution 1 megapixel , frequency 200 hz ) was used . the participants were asked to walk on an 8 m long walkway including two force plates ( type : 9286aa , kistler instrumente ag , winterthur , switzerland ) . the positions of the lower limb segments and the pelvis were determined using the plugingait model ( 16 markers ) with consideration of marker placement error ( szczerbik and kalinowska , 2011 ) . this velocity range was determined during a preliminary study as the natural speed of the walk in young male subjects . angles at the ankle , knee , hip and pelvis in the sagittal , frontal and transversal planes were evaluated at specific gait cycle phases , as determined by force plates and the kinematic system as follows : initial contact ( ic),opposite toe off ( oto),heel rise ( hr),opposite initial contact ( oic),toe off ( to),maximal knee flexion ( mkf),tibia in the vertical position ( tv ) . initial contact ( ic ) , opposite toe off ( oto ) , opposite initial contact ( oic ) , maximal knee flexion ( mkf ) , tibia in the vertical position ( tv ) . ankle movement in the frontal plane ( inversion / eversion ) was not assessed since applying the conventional gait model in the absence of any enhancement in the set of foot markers is not considered reliable for this variable . the kinematic data were processed using standard procedures by vicon nexus and vicon polygon software ( vicon motion systems ltd . , oxford , uk ) . the relationships between angle variables of the lower limbs and the pelvis ( table 1 ) in specific gait cycle phases were determined using statistica software ( version 10.0 , statsoft , inc . , the kolmogorov - smirnov test showed a non - normal data distribution , thus , the spearman correlation coefficient was used . a correlation was considered large for values greater than 0.5 and moderate for values between 0.3 and 0.5 ( cohen , 1988 ) . positive correlations meant that movement in the joints occurred in the same direction ( both for positive or negative ) . for example , a positive correlation between the ankle and the knee in the sagittal plane indicated that increased ankle dorsal flexion was associated with increased knee flexion . conversely , a negative correlation illustrated a contrary joint movement ; for example , in the transversal plane there was internal rotation at the ankle and external rotation at the knee . description of measured angle variables positive positive angle values , negative negative angle values for clarity , our study focused only on adjacent segments and the individual assessment of each plane . all large and moderate ( greater than 0.3 ) correlations ( table 2 ) were statistically significant ( p < 0.05 ) . the highest number of moderate and large correlations was found for the following phases of a closed kinematic chain : heel rise ( 81% ) , opposite toe off and opposite initial contact ( 63% ) . with regard to an open kinematic chain ( maximal knee flexion 38% , 56% ) and borderline phases between closed and open kinematic chains ( initial contact , toe off 50% ) , the number of large and moderate correlations was smaller . the number of large and moderate correlations was slightly higher for the preferred limb ( 61% ) than for the non - preferred one ( 54% ) . in regard to the movement planes , the number of correlations was higher in the sagittal ( 67% ) and transversal planes ( 60% ) compared to the frontal plane ( 36% ) . correlations between joint movements in various gait cycle phases ic initial contact , oto opposite toe off , hr heel rise , oic opposite initial contact , to this combination appears in most of sports movement patterns such as running or throwing , which have to be trained and improved in terms of movement stability . kinematic chain segmental stability appears sequentially dependent , where the inferior segments are significantly correlated to the superior segments . therefore , controlling lower limb segment stability may allow for increased stability at the more proximal segments and , ultimately , the trunk ( graham et al . , 2011 ) . our results also show that segments coupling in relation to the okc and ckc on the example of the gait present relationships that might be generalized for other basic movement patterns used in sports . specifically , if the aim of the coach is to alternate the athletes movement pattern , it should be considered whether to alternate the ckc or okc part of the movement . the same situation also appears when considering the benefits of eliciting changes in the preferred or non - preferred limb . in the sagittal plane , our results showed significant correlations between adjacent segments during the entire stance phase ( a closed chain ) for the knee - hip and hip - pelvis pairs . for the ankle - knee pair , a significant correlation was found only for initial contact , opposite toe off and heel rise . these correlations are subject to changes at the knee joint , what is associated with braking of the body movement and reduced vertical displacement of the centre of mass ( rose and gamble , 2006 ) . during the swing phase ( an open chain ) , significant correlations between the movements of the adjacent segments in the sagittal plane occur as well , however , values of the correlations and the number of the significant ones are smaller . correlations were significant during mkf for the hip - pelvis pair and during tv for the ankle - knee and knee - hip pairs . a significant relationship between movement of the pelvis and the hip in the sagittal plane during a gait cycle was confirmed by franz et al . ( 2009 ) , who found a positive correlation between hip extension and the anterior pelvic tilt . in the frontal plane , the highest values were found during a preswing ( from oic to to ) when the weight was transferred to the contralateral limb and the pelvis tilted towards the ipsilateral limb with associated hip abduction . for the pelvis to return to a neutral position during a midswing ( between mkf and tv ) , simultaneous hip and knee flexion is necessary ( rose and gamble , 2006 ; whittle , 2007 ) . in the transversal plane , a significant correlation between the ankle - knee and knee - hip pairs was observed in almost all gait cycle phases . since rotation in the knee joint is limited , we considered this fact as a correlation between the foot and the hip . ( 2011 ) who revealed a correlation between transverse plane rotation at the foot and transverse plane rotation at the hip and the pelvis . the highest number of moderate and large correlations was found for opposite toe off , heel rise and opposite initial contact . during all of these phases , the lower limb and the pelvis were in a closed kinematic chain . 2012 ) showed significantly greater coupling during the stance phase compared to the swing phase between the movements of the knee joint in the sagittal and transversal planes . although coupling in this study means a movement of one joint in two planes , it suggests that different movements of the lower limbs correlate more in closed kinematic chains than in open ones . the obtained results brought forward some differences in the correlations between the preferred and non - preferred limb . the number of moderate and large correlations was higher for the preferred limb . between the knee and hip movements , the correlations were often larger in the preferred limb in the sagittal plane and larger in the non - preferred limb in the transversal plane . this suggests that functional differences between limbs depend on the contribution of each limb to performance of propulsion and control during able - bodied walking ( michalski et al . , 2011 ; sadeghi et al . , 2000 ) ; the preferred limb appears to be more important for propulsion . some authors have presented correlations between non - adjacent segments and various planes ( svoboda et al . , 2014 ) . however , their studies mostly observed the influence of a foot position on more proximal segments . segments coupling during complex movements depend on both ckc and okc movements , thus , alternation of each movement may change kinematics . despite that therefore , the instructions or interventions focusing on ckc alternation are more effective for movement pattern changes . moreover , the preferred limb initiates kinematics in the direction of propulsion , while the non - preferred limb in internal and external rotation .
aim . to determine the influence of age at onset of type 1 diabetes and of traditional vascular risk factors on the development of diabetic retinopathy , in a cohort of patients who have been followed up after onset . methods . observational , retrospective study . the cohort consists of 989 patients who were followed up after diagnosis for a mean of 10.1 ( sd : 6.8 ) years . the influence of age at diagnosis , glycemic control , duration of diabetes , sex , blood pressure , lipids , bmi , and smoking is analyzed using cox univariate and multivariate models with fixed and time - dependent variables . results . 135 patients ( 13.7% ) developed diabetic retinopathy . the cumulative incidence was 0.7 , 5.9 , and 21.8% at 5- , 10- , and 15-year follow - up , respectively . compared to the group with onset at age < 10 years , the risk of retinopathy increased 2.5- , 3- , 3.3- , and 3.7-fold in the groups with onset at 1014 , 1529 , 3044 , and > 44 years , respectively . during follow - up we also observed an association between diabetic retinopathy and hba1c levels , hdl - cholesterol , and diastolic blood pressure . conclusion . the rate of diabetic retinopathy is higher in patients who were older at type 1 diabetes diagnosis . in addition , we confirmed the influence of glycemic control , hdl - cholesterol , and diastolic blood pressure on the occurrence of retinopathy .	diabetic retinopathy ( dr ) remains a common complication in people with type 1 diabetes ( t1d ) . 39 , 55 , and 84% of t1d patients will develop dr after 10 , 20 , and 40 years of evolution , respectively [ 13 ] . the duration of diabetes and the glycemic control are the risk factors that are most closely related with all forms of dr . other factors such as male gender , hypertension , high body mass index ( bmi ) , nephropathy , dyslipidemia , smoking , and genetic factors appear to influence the onset or progression of dr , although their role is controversial [ 1 , 35 ] . the influence of age at t1d diagnosis on the occurrence of microvascular complications like dr is currently a subject of active debate . some studies have reported that the prepubertal stage , especially the first 5 years of life , might protect from the occurrence of dr [ 68 ] . however , there are authors that point out that such protection disappears as the disease progresses [ 2 , 5 ] , while others have never observed such an effect [ 9 , 10 ] . puberty has a negative influence on the appearance of dr , which is due to the combination of hormonal changes and the poorer control that often accompanies this stage of life . when t1d onset is after age 15 , the literature on the effect of age on dr development is limited and rather confusing . patients aged over 15 at onset have the lowest protection against advanced dr , whereas hietala et al . stated that the risk of proliferative dr ( pdr ) is higher when t1d onset is between 5 and 14 than when it is between 15 and 40 . finally , kullberg et al . reported that the prevalence of dr increases in patients aged 15 to 19 at onset but decreases at onset ages between 30 and 35 . in this study we take advantage of the cohort of patients included in the type 1 diabetes registry of navarra to estimate the risk of dr development according to age at onset and duration of t1d , smoking , blood pressure ( bp ) , bmi , glycemic control as estimated by hba1c levels , and lipid profile . the subjects of the study are all included in the t1d registry of navarra : patients with onset of t1d from january 1990 , who were followed up and treated in the complejo hospitalario de navarra until july 2013 . in all cases , we also measured anti - gad and anti - ia2 antibodies . according to the medical protocol followed , all patients had at least one scheduled outpatient appointment per year . the patients ' data needed for the study were obtained from the electronic health records of the navarra health service . we gathered information about age and sex at onset for all patients . at the screening visit and for every visit through follow - up , we included weight , height , systolic bp ( sbp ) , diastolic bp ( dbp ) , smoking status , and analytical data such as lipid profile and hba1c . when patients had more than one determination of these covariates in a year , we computed the arithmetic mean in the case of continuous variables , while for the categorical ones we chose the value that lasted longest in that year . in all follow - up visits , smoking habits were ascertained and patients were categorized as nonsmokers , ex - smokers , or smokers . a nonsmoker was defined as someone who had smoked fewer than 100 cigarettes in their lifetime ; an ex - smoker was someone who had smoked more than that amount but had quit smoking at least one year before the analysis of data was performed ; finally , a smoker was someone who had not quit smoking or had quit within the last year . bmi was calculated using the formula : weight ( in kilograms ) divided by height ( in meters ) squared . screening and grading for the presence or absence of dr were performed by trained ophthalmologists using fundoscopy in mydriasis at least once every two years , starting five years after diagnosis . in our hospital , t1d patients are always explored by an ophthalmologist . retinal examination by binocular biomicroscopy and a 78/90 d lens was recorded in a standardized format in the electronic health record of the navarra health service . in patients under 12 years retinopathy was graded according to a 5-degree severity scale based on the american academy of ophthalmology 's simplified classification . however , since we had rather few cases of pdr , all grades were grouped together for statistical purposes . from 1990 to 1997 , hba1c was measured using various techniques ( abbott imx , ciba corning glycomet , merck , and menarini hplc ) , but after 1997 , hba1c was determined in all patients with high - performance liquid chromatography ( hplc ; adams a1c ha , menarini diagnostics , florence , italy ; reference range : 4.16.2% ) . in 2005 , the hospital complex obtained level ii laboratory certification of traceability from the diabetes control and complications trial ( dcct ) reference method through the national glycohemoglobin standardization program . hdl - cholesterol and triglycerides ( tg ) were measured by gpo - pap ( roche diagnostics ) . characteristics of the patients at onset of the disease were summarized using frequency and percentages for categorical variables and mean and standard deviations ( sd ) for continuous ones . the cumulative incidence of retinopathy was estimated and graphed for the whole sample and also divided by age groups . 95% confidence intervals based on the cumulative hazard were estimated for 5 , 10 , and 15 years after onset . data were right - censored when no retinopathy event occurred during follow - up or due to loss to follow - up or death . in order to assess the effect of the different variables on retinopathy , firstly , univariate cox - proportional hazards regression models were fitted . we assessed the effect of covariates at onset as fixed effects and complemented the analyses with a dynamic approach that includes the covariates as time - dependent variables , updating the values along the follow - up . the proportionality assumption implicit in the cox models was assessed using weighted residuals and when violated , an interaction term with time was evaluated . the possible modifying effect of age group was evaluated and models were adjusted by age group when appropriate . secondly , a multivariate regression model was fitted with the covariates that had turned out to be significant in the previous step . 989 patients with t1d were followed up from onset , with mean ( sd ) follow - up of 10.1 ( 6.8 ) years . of them , 292 ( 29.5% ) had the onset in childhood ( under 15 years ) and 579 ( 58% ) were men . at onset , 8 ( 0.8% ) had lipid lowering treatment and 12 ( 1.2% ) antihypertensive treatment , figures that increased to 143 ( 14.5% ) and 103 ( 10.4% ) , respectively , at follow - up . antihyperlipidaemic and antihypertensive treatment were more frequent in patients with than without retinopathy ( 20.7% versus 13.5% and 21.5% versus 8.7% , resp . ) . 135 patients developed retinopathy ( 13.7% ) , 121 of whom had nonproliferative retinopathy ( npdr ) and 14 pdr . all patients with pdr had been previously diagnosed with npdr . given the low number of patients with pdr , statistical results are focused on total retinopathy . nevertheless , it deserves to be mentioned that , at onset , patients that develop pdr have similar hba1c mean values compared to the rest of the patients ( 10.9 ( 4.1 ) versus 11.0 ( 2.5 ) ) , but at follow - up , patients that developed pdr had hba1c mean values of 9.39 ( 2.01 ) , whereas those that developed npdr had 8.30 ( 1.51 ) , and those with no retinopathy had 7.74 ( 1.33 ) . as expected , the cumulative incidence increased over time during the course of diabetes . it was very low during the first 5 years after onset , but it undoubtedly increased after 10 and , especially , 15 years . we observed that the rate of retinopathy was higher in patients that were older at diagnosis . the highest increase was observed in the group whose onset was at age 45 years . notably , after 15 years of follow - up its cumulative incidence was 12 times higher than that observed in the group of patients whose onset was at age < 10 years ( table 2 and figure 1 ) . in the univariate analysis , male gender , smoking , sbp , and hdl - cholesterol at onset however , the association of hba1c with dr development was only marginally significant ( p = 0.079 ) . remarkably , when taking the patients who were younger than 10 at onset as the reference group , the risk increased according to age at onset ( table 3 ) . when an additional univariate analysis was performed for each variable according to its evolution along the follow - up period , a significant association with dr development was again observed for smoking , sbp , and hdl - cholesterol , with hrs ( 95% ci ) similar to the former ones . unlike what happened when the analysis was performed using values at onset , dbp , triglycerides , bmi , and hba1c were now significantly associated with dr . finally , the association between ldl - cholesterol and dr was slightly above the limit of statistical significance [ 1.06 ( 1.00 , 1.13 ) , hr ( 95% ci ) , p = 0.052 ] ( table 4 ) . the multivariate analysis confirmed the trend observed in the univariate one and the risk of developing dr increased according to age at onset , albeit less markedly ( table 5 ) . in any case , when focusing on pediatric age , children with onset at the peripubertal period exhibited a significantly higher risk when compared with those < 10 , the hr increasing more than 2-fold . in adult patients , the risk of dr development increased more than three times with respect to children < 10 , and , notably , it was almost 4 times higher in the group of patients 45 at onset [ hr 3.78 ( 95% ci : 1.3710.41 ) ] . the multivariate approach confirmed that , among the time - dependent variables , dr development along follow - up was also significantly influenced by dbp , hdl , and hba1c , the latter when values were above 9% ( table 5 ) . the rate of dr increases with the age of diagnosis . taking advantage of the follow - up of the patients included in the t1d registry of navarra , we have confirmed the influence of some controversial risk factors on the occurrence of dr . remarkably , we describe for the first time that the risk of dr 15 years after diagnosis increases with increasing age of onset , being highest in those with onset at age 45 . hba1c is the factor exhibiting the highest impact on the development of dr . in the wisconsin study , the hr ( 95% ci ) to develop dr for patients with hba1c from 9.5 to 10.5 , compared with patients with hba1c levels < 9.5% , is 1.72 ( 1.34 , 2.21 ) . however , it rises to 2.41 ( 1.91 , 3.06 ) when hba1c ranges from 10.6 to 12% and is even higher , 3.65 ( 2.87 , 4.65 ) , for hba1c values > 12% . we can not compare these results with ours , because our highest quartile of hba1c is lower than the lowest of theirs , but both studies are consistent in showing the relationship between poor metabolic control , as measured by glycated hemoglobin , and dr development and progression . this relationship has been shown in several publications based on national or regional t1d records , some of them recently [ 1 , 18 ] . t1d duration is the other factor clearly related to the onset and progression of dr [ 3 , 17 ] , and in our series this association was also observed . regarding the factors most frequently discussed , our results match those published on the risk of dr [ 1 , 3 , 17 ] , which is higher in men than in women , although this difference disappears when the effect of confounding variables is prevented by the multivariate analysis , suggesting that the risk is due to factors other than gender . the relationship between bp and dr has been generally accepted , but results have not always coincided . published data are influenced by the type of analysis and are divergent if analysis of bp is performed at baseline or throughout follow - up , if bp is stratified by ranges or by 10 mmhg increments , or if the comparison is only between hypertensive and normotensive patients [ 3 , 17 ] . different results have also been reported depending on how dr is evaluated : as a whole , or taking npdr and pdr cases separately , or even if the progression from one to the other is analyzed . our results largely match those of the wisconsin study , although the latter treats npdr and pdr as different entities and we have analyzed them in a single group since there were only 14 patients with pdr in the t1d registry of navarre . our results are in accordance with those of the linkoping diabetes complications study in that although smoking is associated with the risk of dr in the univariate analysis , there is no relationship between both variables after adjusting for confounding factors in the multivariate analysis . therefore , there must be other factors closely associated with smoking to explain this recurrent finding . the fact that smoking is linked with a worse glycemic control may be responsible for such an observation . dyslipidemia , especially increased triglyceride and decreased hdl - cholesterol levels and , to a lesser extent , the increase in total cholesterol and ldl - cholesterol , is a risk factor , albeit weak , for the occurrence of dr , especially in its severe forms [ 22 , 23 ] . thus , additional factors ( genetic , inflammatory , and metabolic ) might be necessary for lipids to induce such effect . in any case , there is experimental evidence that increased and biochemically altered plasma lipids lead to cytotoxicity in retinal capillary cells . in our study the findings on ldl- and hdl - cholesterol and triglycerides ( tables 3 , 4 , and 5 ) are consistent with the data published on a finnish population . the occurrence of dr ( pdr plus ndrp ) has been related to high bmi in the dis study in sweden , but , in finland , hietala et al . our results , in the multivariate analysis , differ from those obtained in sweden , perhaps due to a smaller number of events in our series ( 135 versus 247 patients ) . the influence of age of diagnosis on the onset and progression of dr has been the subject of numerous publications ; most of them focused on children and the probable protective effect of the prepubertal period . ours is the first study to include patients who have been diagnosed with t1d over the age of 40 . in our series , patients within the pubertal period , that is , children aged 1014 years , exhibit a risk of developing dr which is 2.5 times higher than the risk associated with children who are aged 09 years . these data are consistent with the results of most authors [ 58 ] , especially those of olsen et al . , but differ from those obtained by holl et al . and by the wisconsin study . there is a controversy about the effect of hyperglycemia on the occurrence of microvascular complications before puberty . discrepancies may be due to different interpretations about what is the exact range of age encompassed by the term puberty , which have led to heterogeneous ways of grouping patients aged < 15 years . there is a real need to gain knowledge on this topic to take advantage of the age at diagnosis to help in deciding the extent of the aggressiveness of insulin treatment . during puberty , glycemic control is worsened and the risk of microangiopathy development increases , which is partly due to insulin resistance . the insulin sensitivity in the middle of puberty is reduced by 3035% compared to the late stage of puberty , prepubertal childhood , or adulthood . this seems to be mainly due to the effects of the growth hormone ( gh ) and the insulin growth factor-1 ( igf-1 ) . the actions of gh are mediated by igf-1 , and the levels of both molecules have been correlated with the thickness of the capillary basement membrane as well as with diabetic angiopathy . furthermore , the increased levels of sex hormones are directly related to vascular structural abnormalities associated with diabetes complications . this is due to the ability of such molecules to increase polyol metabolism in the basement membrane , as has been demonstrated in animal models . there is an association between capillary basement membrane thickening and the duration of t1d in the postpubertal period . in fact , diabetes control markers , such as hba1c and fasting glucose , are positively correlated with thickness in postpubertal but not in prepubertal patients , thus suggesting an interaction between diabetes control and puberty . psychosocial factors associated with adolescence may also contribute to a poorer glycemic control during this phase and thus to its subsequent undesirable effect on morbidity in patients with t1d . our patients aged 15 to 29 years have a risk of dr which is 3-fold the risk exhibited by children < 10 . not surprisingly , these results are again in agreement with those obtained by olsen et al . from the danish study group of diabetes in childhood and in disagreement with those recorded in the wisconsin study . in our series , the risk continues to increase with age , so that it is even higher for patients > 30 and , especially , almost 4-fold , for patients 45 . hammes et al . also found an increased risk for pdr in patients diagnosed with t1d and aged 15 to 40 years . by contrast , kullberg et al . , studying patients aged up to 36 at onset and also gathering all forms of retinopathy , observed a decrease in the prevalence of dr in patients older than 30 . in the finnish records , the risk of pdr was lower in patients who were aged 15 to 40 at onset compared to those whose onset was at puberty . the beta cells are deemed to be best preserved when diabetes onset is in adulthood . as a consequence , the glycemic control would be easier when development of t1d was at an age long after puberty , and this would explain the lower risk of dr found in these patients . our group had previously published that , in the patients included in the t1d register of navarra , a relationship between the age of onset and glycemic control also exists . however , in our cohort the worst glycemic control at follow - up was observed in the group of oldest patients at t1d onset . thus , from this point of view our present findings about age at onset and risk of dr are consistent with our previous observations . the main one is that the number of patients who developed pdr was rather small in our cohort . for this reason , we were not able to analyze separately npdr and pdr ; that is , we do not have information about whether or not there are risk factors that influence differently the progression to one entity or to the other . we are confident that longer follow - up , lasting over 20 years , will allow us to obtain more information in the future . , we want to remark that our cohort includes a significant number of patients who were followed up from diagnosis . in fact , the onset of disease of each patient is the time of his / her inclusion in the study , which allows the analyses of incidence at different times , that is , 5 , 10 , or 15 years , to be more accurate than if the onset and baseline had not matched . furthermore , we consider that the risk of bias that could have led to inaccurate results is rather low since the follow - up of all patients took place at the same hospital , and the screening and grading for the presence of dr were always performed by the same ophthalmologists , who followed similar clinical criteria throughout the study . in sum , the analysis of the t1d registry of navarra shows , for the first time , that there is a relationship between the age at t1d diagnosis and the prevalence of dr throughout life and confirms the influence of t1d duration and glycemic control on the appearance of dr . our results also support the view that bp and lipid profile have an effect on dr .
in the contemporary era , use of drugs is the dominant paradigm of health care . the most quotidian drug used for fever and pain is paracetamol . although adverse reactions to paracetamol in india are rare , at times they can cause life - threatening situations . stevens - johnson syndrome ( sjs ) is one such potentially lethal adverse drug reaction . the most reported cases of analgesic - induced sjs were due to oxicams or propionic acid derivatives . there are very few detailed reports of sjs due to the use of paracetamol . we report a case of sjs , which occurred due to the use of paracetamol . the clinical features of this condition and multidisciplinary management of the patient are described in brief .	steven - johnson syndrome ( sjs ) is an infrequent and a severe form of erythema multiforme ( em ) . it can occur due to an adverse hypersensitivity reaction to drugs which results in skin and mucosal eruptions that can be potentially fatal . it is considered to be a less severe form of toxic epidermal necrolysis ( ten ) . the only difference being the extent of epidermal detachment ; that is , 30% of the total body surface area ; while 1030% is known as sjs - ten overlap . a new eruptive fever with stomatitis and ophthalmia was described as a severe variant of em and was termed by steven and johnson in 1922 . sjs may present as a nonspecific febrile illness leading to malaise , headache , cough , rhinorrhea with polymorphic lesions of the skin and mucous membrane characterized by acute blisters and erosions . the incidence of sjs has been estimated to be around 16/1,000,000 persons per year with a mortality rate of 15% which rises up to 30% in ten . multiple drugs have been identified to cause sjs and ten , antibiotics ( sulfonamides ) being the most common . the drugs that cause sjs commonly are antibacterials ( sulfonamides ) , anticonvulsants ( phenytoin , phenobarbital , and carbamazepine ) , nonsteroidal anti - inflammatory drugs ( oxicam derivatives ) , and oxide inhibitors ( allopurinol ) . paracetamol is among the most extensively used analgesic and anti - pyretic because of easy availability and cost - effectiveness . despite being considered relatively safe , very few cases of em or sjs have been reported with the ingestion of paracetamol . publications from 1995 to 2011 describing sjs and ten in indian population were searched by patel et al . in pubmed , medline , embase , and uk pubmed central electronic databases showed 6.17% of cases of sjs and ten were due to the ingestion of paracetamol . hence , we present a rare case of sjs occurred due to the ingestion of paracetamol . a 14-year - old male patient reported to department of oral medicine and radiology with a chief complaint of painful ulceration of lips and oral cavity leading to difficulty in opening mouth and eating food since 5 days . history of present illness revealed that there was burning sensation followed by ulcers which appeared first in oral cavity , lips , and other parts of body including chest , arms , legs and thighs , and genital organs [ figure 1a and b ] . the redness of eye was evident , and there was a history of watery discharge . numerous healed lesions were also seen on the chest , axilla which gave the typical appearance of target lesions or bull 's eye appearance [ figure 1c ] . ( a ) initial presentation of the case showing encrusted lesion on lips and intra - oral erythema . ( b ) hemorrhagic erosion on mucous membrane of glans penis , ( c ) targetoid rashes over chest the past medical history revealed that the patient was suffering from fever and pain since 2 weeks . the patient was prescribed tablet crocin for fever and pain for 7 days by a local medical practitioner . the patient was relieved from fever and pain but later he had burning sensation followed by ulcers in the oral cavity and extra - oral surface . intra - oral examination revealed a solitary mixed red and white lesion present on both right and left buccal mucosa . on left buccal mucosa it measured 10 mm 8 mm in diameter and right side about 16 mm 9 mm approximately . the lesions have well - defined borders and on palpation it was non scrapable and nontender with a rough texture . the upper and lower lips were swollen , and hemorrhagic crusts were present along with profuse bleeding . laboratory investigations revealed leukocytosis ( white blood cells , 15101/l , reference value , 400011,000/l ) and raised c - reactive protein 59.87 g / ml ; reference range , 05 g / ml . we had subjected the patient to only the hematological investigation as the lesion being acute , and the patient was under severe discomfort . based on clinical examination , medical history , and physical examination our diagnosis was sjs . the patient was treated under an expert guidance of dermatologist with systemic steroids ; tablet prednisolone 30 mg twice daily daily for 7 days . further reduced to 20 mg twice daily for next 7 days . gradually , 10 mg and 5 mg for consecutive 7 days were administered . application of kenacort ( triamcinolone ) ointment thrice a day was advised for ulcers in the oral cavity . recall after 2 weeks revealed almost resolved lesions on all the surfaces and completely recovered approximately in 40 days [ figure 2a and b ] . ( a ) healing of crusting of lips and healing lesion of buccal mucosa , ( b ) healed lesion of upper and lower lips sjs is an uncommon , severe , mucocutaneous blistering disorder with an acute and unpredictable onset causing considerable morbidity . previously , sjs was considered as em major , but now is considered distinct from em on the basis of severity , presence of constitutional signs , atypical target lesions with tendency to confluence , positive nikolsky 's sign , more than one mucosal site involvement , and residual sequelae . in the oral cavity , sjs causes widespread ulcerative lesions . prodromal symptoms seen in about 30% of cases and may possibly initiate within 13 weeks of starting a new drug , and lasts for 12 weeks , presenting with flu - like symptoms , sore throat , headache , arthralgias , myalgias , fever , and other rashes . ocular changes such as dry eyes that resemble those of mucous membrane pemphigoid may be noted in few cases . our patient did not report any prodrome , but the eye and genital ulcerations were present , along with skin and mouth ulcers . although many factors have been proposed as risk factors of sjs , including drug - induced , infections , malignant disorders , and graft rejection , most of them were due to the adverse effect of drugs . the most common drugs are a nonsteroidal anti - inflammatory drug ( nsaids ) , antipsychotics , antibiotics , allopurinol , and anticonvulsants . sjs can be differentiated from other skin conditions on three clinical criteria , ( i ) the pattern of individual skin lesions , ( ii ) distribution of lesions , and ( iii ) extent of epidermal detachment . the characteristic findings in sjs are widespread erythematous or purpuric macules which form flat atypical target lesions as the disease progresses to cause full thickness epithelial necrosis . our case showed ulceration of oral cavity , crusting of lips and profuse bleeding , involvement of eye with redness and watery discharge , ulceration of genital region along with numerous healed lesions on chest , axilla which showed typical appearance of target lesions or the lesions were widespread as compared to em , which is localized . in a study done among children in the hospital , it was found that anticonvulsant drugs were reported the highest risk for sjs and paracetamol lest and vaccines presented no risk at all . among the nsaids , paracetamol and nimesulide the severe cutaneous adverse reactions ( scars ) study has found an overall risk of sjs with oxicam derivatives . it reports the increased risk with paracetamol from germany , italy , and portugal except france but very few cases from india . however , paracetamol is found to be a potential risk factor in children according to survey data from pediatric patients from the scar . the present case was diagnosed as paracetamol induced sjs based upon the fact that a sequential relationship with the drug was established , and correlation with exposure with signs and symptoms was made . reported a case of acetaminophen induced sjs and ten with widespread macula - papular rash , stinging in the eyes , oral mucosal ulcerations , and high - grade fever . similar features were seen in our case , but there was the absence of high - grade fever and epithelial detachment . the first step in the management was an immediate withdrawal of the offending agent followed by supportive care . garcia - doval et al . , report that earlier the drug is withdrawn , better the prognosis while exposure to drugs with longer half - lives increases the risk of death . routine antibiotics are not indicated unless there is the evidence of infection as fever may be part of the disease process . however , in our case , there were lesions on axilla , abdomen , thighs and trunk region in the healing phase , so debridement was not a necessary step . topical antiseptics ( 0.5% silver nitrate or 0.05% chlorhexidine ) are used to paint , bathe , or dress the patients . dressings may be gauzes with petrolatum , silver nitrate , povidone - iodine , and hydrogels . some authors use biologic skin covers after epidermal stripping cadaveric allografts , cultured human allogeneic or autologous epidermal sheets . in our case , dramatic improvement in both sjs - ten has been reported with the use of intravenous immunoglobulin , 0.20.75 g / kg body weight . alternative systemic treatment methods for the acute phase of sjs - ten include hemodialysis , plasmapheresis , cyclophosphamide , and cyclosporine . , their use can lead to delayed wound healing , increased chances of infection , masking of early signs of sepsis , gastrointestinal bleeding , and increased mortality . if steroids are to be used , it should be initiated during initial stage and rapidly tapered off . hence , we prescribed tablet predinsolone 30 mg three times daily daily for 7 days . gradually , 10 mg and 5 mg for consecutive 7 days . his condition improved no sequelae were found during 3540 days of follow - up . this case report reports the fact that severe hypersensitivity reactions can occur with paracetamol , which can be possibly dangerous and life - threatening . the authors have obtained the necessary patient consent forms where the patients have given their approval for participation in the investigation , followed by representation in the concerned article . the patients do understand that the authors will ensure that their identities wo n't be revealed , however anonymity can not be guaranteed . the authors have obtained the necessary patient consent forms where the patients have given their approval for participation in the investigation , followed by representation in the concerned article . the patients do understand that the authors will ensure that their identities wo n't be revealed , however anonymity can not be guaranteed .
children born at term with low birth weight ( lbw ) are regarded growth restricted and are at particular risk of adverse health outcomes requiring a high degree of parental participation in the day - to - day care . this study examined whether their increased risk of special health care needs compared to other children may influence mothers opportunities for participation in the labor market at different times after delivery . data from 32,938 participants in the population - based norwegian mother and child cohort study with singleton children born at term in 20042006 were linked to national registers in order to investigate the mothers employment status when their children were 13 years in 2007 and 46 years in 2010 . children weighing less than two standard deviations below the gender - specific mean were defined as lbw children . although not significantly different from mothers of children in the normal weight range , mothers of lbw children had the overall highest level of non - employment when the children were 13 years . at child age 46 years on the other hand , lbw was associated with an increased risk of non - employment ( rr 1.39 : 95 % ci 1.111.75 ) also after adjustment for factors associated with employment in general . in accordance with employment trends in the general population , our findings show that while mothers of normal birth weight children re - enter the labor market as their children grow older , mothers of lbw children born at term participate to a lesser extent in paid employment and remain at levels similar to those of mothers with younger children .	improved neonatal care has contributed to increased survival among preterm and low birth weight ( lbw ) infants [ 1 , 2 ] . however , the risk for chronic conditions requiring a high degree of parental participation in the day - to - day care increases markedly with decreasing birth weight and gestational age [ 36 ] . while lbw children ( i.e. , < 2.500 g ) born preterm can be appropriate , constitutional , or pathological small for their gestational age ( sga ) , most lbw children born at term are sga and many have failed to reach expected growth in utero and are regarded growth restricted [ 7 , 8 ] . the risk of perinatal morbidity and mortality increases the longer the slow growth has occurred [ 9 , 10 ] , and growth restricted children are at particular risk of severe intellectual disabilities and cerebral palsy , even exceeding the risk found among children born preterm [ 1113 ] . the severity and chronicity of many conditions prevalent in these children can have a profound effect on their health and functioning , and many are identified as having special health care needs ( shcn ) [ 1416 ] . these children s care needs are often of a type and amount beyond that required by children of the same age in general , and can therefore , as primary caregivers , particularly impinge on mothers time budgets and opportunities for regular participation in the labor market [ 1719 ] . mothers of children with shcn often report difficulties engaging in paid work , and more often than mothers in general to have missed days from work , to have cut work hours , or left paid employment altogether due to their child s additional care needs [ 20 , 21 ] . however , the association between child care and work participation is complex and dependent on factors related to the child ( e.g. , age ) , the mother herself ( e.g. , educational attainment ) , and the household at large ( e.g. , financial circumstances ) . mothers in households that can manage on one salary may prefer to stay at home or to reduce work hours while their children are young , before re - entering or increasing their work participation sometime as their children reach preschool and school age [ 23 , 24 ] . this typical pattern characterized by increasing work hours with increasing child age is not as apparent among mothers of children with shcn . although lbw is associated with excess risk of morbidity and shcn [ 4 , 5 ] , limited knowledge exists about how the care needs of lbw children born at term can influence their mothers opportunities for participation in the labor market . through linkage of population - based data from national registers , this study aimed to investigate associations between children s birth weight and mothers risk of non - employment during preschool years . in order to examine employment trends as children age , we investigated mothers employment status when the children were 13 years , and again when the children were 46 years of age . as the risk of non - employment has been found to be most pronounced among mothers of children with severe care needs [ 21 , 22 ] , we expect the risk to be increasing with decreasing birth weight . moreover , due to the adverse health outcomes observed among many lbw children and as women in general increase their work participation as children age , we expect the association between the child s birth weight and maternal work participation to become more pronounced as the child grows older . the study population comprised participants in the population - based norwegian mother and child cohort study ( moba ) , conducted by the norwegian institute of public health . the study recruited pregnant women at their first routine ultrasound examination around weeks 1718 of gestation from 1999 to 2008 , and includes about 107,000 pregnancies in total . the moba cohort is linked to the medical birth registry of norway , which is based on compulsory notification of all deliveries in norway with more than 12 completed weeks of gestation . the registry contains the national identification number for all participants in the study , allowing linkage with the central population register , the national education database of statistics norway , and the employment register in the national insurance administration . this linkage provided longitudinal data with annual updates for both mothers and children through 2010 . for the purpose of this study , mothers with children born in 20042006 were selected , of whom 43.5 % of those invited consented to participate in the moba study . since several thousand women have participated with more than one pregnancy ( > 15 % ) , and as participation in the study was linked to national registers , only unique pairs of mothers and children were eligible . altogether , data for 40,502 women were successfully linked to the registries . among these cases , twin and triplet births ( 1.7 % ) and deliveries prior to week 37 and after week 41 of gestation , were excluded ( 12.9 % ) , leaving 34,587 cases eligible for the study . among the eligible cases , we excluded cases where the mother had emigrated or where either the mother or the child had died ( 1.9 % ) , children born with severe congenital malformations ( 2.6 % ) , and cases for which data on birth weight or gestational age were missing ( 0.3 % ) , leaving a sample of 32,938 mothers and children who were residents of norway in 2010 . the study was approved by the regional committee for medical research ethics in south - eastern norway . employment status is reported annually by employers and recorded in the employment register in the national insurance administration . as the total labor force constitutes the sum of all employed and unemployed persons , non as conceptualized in this study , non - employment is therefore defined as both the unemployed and those outside the labor force due to other reasons than unemployment . the proportion of women in the labor force of comparable age as the study participants age in 2007 ( m 31.9 : sd 4.7 ) was in the range of 8285 % in 2007 , and 8186 % 3 years later in 2010 . at the same time , the general unemployment rates among women were low , with 2.5 and 3.0 % unemployment in 2007 and 2010 , respectively [ 29 , 30 ] . the medical birth registry of norway provided data on birth weight , gestational age , year of birth , child s sex , parity , and mother s age and marital status at childbirth . birth weight was standardized for gender into z - scores with a mean birth weight of zero applying data from all singleton live births in the total moba cohort . mean birth weight was 3,535 g ( sd 548 g ) for girls and 3,660 g ( sd 573 g ) for boys . a one unit decrease in the z - score equals a decrease in birth weight of one standard deviation , and children weighing less than two standard deviations below the mean were defined as lbw children ( boys < 2.510 g : girls < 2.436 g ) , all of whom were sga below the 10th percentile according to norwegian birth weight by gestational age standards . children with birth weights ranging from the mean to one standard deviation above the mean were selected as referents . data on educational attainment was obtained from the national education database of statistics norway according to a six - digit code reflecting nine discrete levels of education . the women s highest level of education at childbirth was categorized as educational attainment below high school graduate ( 03 ) , high school graduate ( 45 ) , lower college or university level ( 6 ) , and higher college or university level , including postgraduate levels ( 78 ) . an indicator of the household s dependency on the mother s income was obtained from questionnaire assessments provided by the participants in the moba study about week 17 of gestation . the women were asked is it possible for your household to manage financially without your income ? , and we considered responses from women reporting without difficulties as households not dependent on the mothers income . as there is considerable variation in work participation among mothers of toddlers and preschool children , we first investigated the mothers risk of non - employment in 2007 when their children were 13 years , and again in 2010 when their children were 46 years of age , adjusted for several factors associated with maternal employment in general . the child s age was constructed as the difference between the year of follow - up and the child s birth year . analyses were performed using stata / se version 12.1 and as non - employment is common among women with small children , robust poisson regression analyses were performed . results are presented as adjusted risk ratios ( rr ) with corresponding 95 % confidence intervals ( ci ) . apart from the child s birth weight , the models included gestational age coded according to conventional cut - offs as early term ( 3738 weeks ) and full term ( 3941 weeks ) births , mother s age , educational attainment , parity , and household type , all according to characteristics at childbirth , in addition to self - reported household dependency on own income and the child s age at follow up in 2007 and 2010 , respectively . the study population comprised participants in the population - based norwegian mother and child cohort study ( moba ) , conducted by the norwegian institute of public health . the study recruited pregnant women at their first routine ultrasound examination around weeks 1718 of gestation from 1999 to 2008 , and includes about 107,000 pregnancies in total . the moba cohort is linked to the medical birth registry of norway , which is based on compulsory notification of all deliveries in norway with more than 12 completed weeks of gestation . the registry contains the national identification number for all participants in the study , allowing linkage with the central population register , the national education database of statistics norway , and the employment register in the national insurance administration . this linkage provided longitudinal data with annual updates for both mothers and children through 2010 . for the purpose of this study , mothers with children born in 20042006 were selected , of whom 43.5 % of those invited consented to participate in the moba study . since several thousand women have participated with more than one pregnancy ( > 15 % ) , and as participation in the study was linked to national registers , only unique pairs of mothers and children were eligible . altogether , data for 40,502 women were successfully linked to the registries . among these cases , twin and triplet births ( 1.7 % ) and deliveries prior to week 37 and after week 41 of gestation , were excluded ( 12.9 % ) , leaving 34,587 cases eligible for the study . among the eligible cases , we excluded cases where the mother had emigrated or where either the mother or the child had died ( 1.9 % ) , children born with severe congenital malformations ( 2.6 % ) , and cases for which data on birth weight or gestational age were missing ( 0.3 % ) , leaving a sample of 32,938 mothers and children who were residents of norway in 2010 . the study was approved by the regional committee for medical research ethics in south - eastern norway . employment status is reported annually by employers and recorded in the employment register in the national insurance administration . as the total labor force constitutes the sum of all employed and unemployed persons , non as conceptualized in this study , non - employment is therefore defined as both the unemployed and those outside the labor force due to other reasons than unemployment . the proportion of women in the labor force of comparable age as the study participants age in 2007 ( m 31.9 : sd 4.7 ) was in the range of 8285 % in 2007 , and 8186 % 3 years later in 2010 . at the same time , the general unemployment rates among women were low , with 2.5 and 3.0 % unemployment in 2007 and 2010 , respectively [ 29 , 30 ] . the medical birth registry of norway provided data on birth weight , gestational age , year of birth , child s sex , parity , and mother s age and marital status at childbirth . birth weight was standardized for gender into z - scores with a mean birth weight of zero applying data from all singleton live births in the total moba cohort . mean birth weight was 3,535 g ( sd 548 g ) for girls and 3,660 g ( sd 573 g ) for boys . a one unit decrease in the z - score equals a decrease in birth weight of one standard deviation , and children weighing less than two standard deviations below the mean were defined as lbw children ( boys < 2.510 g : girls < 2.436 g ) , all of whom were sga below the 10th percentile according to norwegian birth weight by gestational age standards . children with birth weights ranging from the mean to one standard deviation above the mean were selected as referents . data on educational attainment was obtained from the national education database of statistics norway according to a six - digit code reflecting nine discrete levels of education . the women s highest level of education at childbirth was categorized as educational attainment below high school graduate ( 03 ) , high school graduate ( 45 ) , lower college or university level ( 6 ) , and higher college or university level , including postgraduate levels ( 78 ) . an indicator of the household s dependency on the mother s income was obtained from questionnaire assessments provided by the participants in the moba study about week 17 of gestation . the women were asked is it possible for your household to manage financially without your income ? , and we considered responses from women reporting without difficulties as households not dependent on the mothers income . as there is considerable variation in work participation among mothers of toddlers and preschool children , we first investigated the mothers risk of non - employment in 2007 when their children were 13 years , and again in 2010 when their children were 46 years of age , adjusted for several factors associated with maternal employment in general . the child s age was constructed as the difference between the year of follow - up and the child s birth year . analyses were performed using stata / se version 12.1 and as non - employment is common among women with small children , robust poisson regression analyses were performed . results are presented as adjusted risk ratios ( rr ) with corresponding 95 % confidence intervals ( ci ) . apart from the child s birth weight , the models included gestational age coded according to conventional cut - offs as early term ( 3738 weeks ) and full term ( 3941 weeks ) births , mother s age , educational attainment , parity , and household type , all according to characteristics at childbirth , in addition to self - reported household dependency on own income and the child s age at follow up in 2007 and 2010 , respectively . a total of 22.8 % of the mothers were not employed when their children were 13 years in 2007 , dropping to 15.6 % when their children were 46 years in 2010 . mothers of the youngest children were less likely to be employed during early motherhood , while the child s age influenced maternal employment status less when the children were 46 years . a trend towards lower work participation with decreasing child birth weight was evident , and the proportion of mothers not in paid employment was considerably higher across the whole birth weight distribution when the children were 13 years as compared to when the children were 46 years ( table 1 ) . although mothers of lbw children had the overall highest level of non - employment when their children were 13 years ( 25.3 % ) , lbw was not independently associated with an increased risk of non - employment during early motherhood . non - employment was common also among mothers of children with birth weights above the mean during this period ( 22.0 % ) , and factors such as low educational attainment , young age , and single - motherhood were more important for maternal employment status when the children were 13 years . however , when the children were 46 years , lbw was significantly associated with the mothers employment status ( crude rr 1.65 : 95 % ci 1.312.08 ) . lbw had a substantial independent effect on maternal employment status also after adjusting for other important factors such as educational attainment , single - motherhood , and non - dependence on the mother s income ( rr 1.39 : 95 % ci 1.111.75 ) . among mothers of lbw children , 24.0 % were not employed when their children were 46 years as compared to 14.6 % among mothers of children with birth weights ranging from the mean to one standard deviation above the mean . as compared to their own employment levels when the children were 13 years , mothers of lbw children remained at a high level of non - employment with a relative decrease of only 1.3 % points during this period , in contrast to the referents that had a relative decrease of 7.4 % points . in fact , their level of non - employment at child age 46 years remained quite similar to that of mothers of young children aged 13 in the normal weight range.table 1adjusted risk ratios for non - employment at two different times after delivery among mothers of children born at term in norway during 20042006numberchild s age 13 years ( 2007)child s age 46 years ( 2010)% not employedrr95 % ci% not employedrr95 % citotal32,93822.815.6 birth weight > 1.00 sd above mean4,13422.21.010.951.0814.50.990.911.070.000.99 sd above mean12,66022.01.00reference14.61.00reference0.011.00 sd below mean12,79723.41.030.981.0816.51.040.951.141.012.00 sd below mean3,11423.71.010.951.0916.71.091.021.14<2.00 below mean23325.31.010.811.2724.01.391.111.75 gestational age full term ( weeks 3941)26,82922.71.00reference15.31.00referenceearly term ( weeks 3738)6,10923.00.970.931.0317.01.040.971.10 mother s age 24 years and younger4,03237.91.541.451.6325.21.311.211.412529 years11,06821.61.131.071.1814.81.101.041.1730 years and older17,83820.11.00reference13.81.00reference educational attainment < high school graduate2,80045.12.021.872.1736.22.852.593.14high school graduate7,44427.61.331.241.4320.81.761.611.93lower college / university17,33218.30.960.901.0211.41.020.931.11higher college / university5,36218.91.00reference11.11.00reference parity 1st16,33323.81.00reference15.71.00reference2nd10,49219.70.940.901.0014.10.970.911.033rd4,79723.51.131.061.2016.31.091.001.184th or higher1,31632.21.411.291.5423.01.341.201.49 household type two - parent31,63421.91.00reference14.91.00referencesingle - mother1,30445.21.551.451.6632.01.561.441.70 dependency on mother s income household dependent27,48121.51.00reference14.81.00referencehousehold not dependent5,45729.41.411.351.4819.51.381.301.46 child s age in 2007 and 2010 1/4 years11,37926.81.351.291.4216.21.121.061.192/5 years10,68320.81.030.981.0815.21.020.961.083/6 years10,87620.51.00reference15.21.00referenceall categories according to characteristics at childbirth , except child s age which represents age at follow - up in 2007 and 2010 adjusted risk ratios for non - employment at two different times after delivery among mothers of children born at term in norway during 20042006 all categories according to characteristics at childbirth , except child s age which represents age at follow - up in 2007 and 2010 this study shows that there is a lasting association between children s birth weight and mothers employment status during preschool years , and that the risk of non - employment generally increases with decreasing birth weight . the excess morbidity and elevated care needs observed in lbw children born at term appear to influence mothers opportunities for participation in the labor market for an extended period of time . we observed a negative association between birth weight and work participation becoming more pronounced as the child grows older , also after adjustment for important factors associated with employment in general . although a somewhat smaller proportion of mothers of lbw children were out of paid employment as their children age , the employment levels among mothers of children in the normal weight range increased markedly during this period , corresponding well to employment trends in the general norwegian population . mothers of lbw children , on the other hand , remained at levels similar to those of mothers with young children in the normal weight range . the association observed between birth weight and work participation likely reflects a higher rate of health problems and disability in lbw children born at term , indicating that many children in the lower weight range require parental care and supervision above that needed by children of comparable age within the normal weight range . our findings thus correspond with studies showing elevated levels of shcn among children in the lower weight range [ 46 ] , and mirror studies showing a trend towards lowered maternal work participation in accordance with the severity of the child s condition [ 21 , 22 ] . to our knowledge , this is the first study to investigate associations between children s birth weight and mothers employment status . although a number of studies have investigated associations between children s shcn and mothers work participation , previous studies have almost exclusively utilized cross - sectional study designs , been based on parental report , or lacked appropriate reference groups of children without shcn [ 25 , 35 ] . using a large population - based sample and register - based data to longitudinally investigate the risk of non - employment the observed employment trends suggest that birth weight constitutes a valid indicator of children s care needs in early life in which other objective data of children s shcn can be hard to obtain . because the impact of different conditions varies accordingly , a child s care needs must be considered in the context of the child s stage of development . thus , in order to examine the relative effect of caring for a child with special needs , it is important to contrast the work participation of mothers of children with shcn with that of mothers in general with children of comparable age . for instance , our finding that children s birth weight affects mothers work participation to a larger extent as children age contradicts conclusions drawn in studies utilizing samples solely of children with shcn [ 21 , 22 ] . although mothers of lbw children had the overall highest level of non - employment when the children were 13 years , reduced work participation during early motherhood is common also in the general population . norwegian figures of female labor market participation show that approximately 1 in 4 women with small children are non - employed , dropping to 1 in 5 as children grow older . with close to 90 % of children in kindergarten and as norwegian unemployment rates are low and well below the oecd average , most non - employed women are out of paid employment due to other reasons than lack of child care and unemployment . thus , because many women are not employed for shorter and longer periods when their children are young , elevated levels of non - employment among mothers of children with shcn need not solely reflect the additional care needs of these children , but rather a general trend towards lowered work participation during early motherhood . as we have shown , other important factors such as educational attainment and household income greatly influence on women s employment choices , irrespective of their children s care needs although a consistent trend towards higher risk of non - employment with decreasing birth weight is evident , some caution is warranted due to a relative small number of lbw children . the response rate in moba is also lower than optimal , and although not uncommon for large epidemiological studies , self - selection to the study may result in deviations from the larger population from which the women were sampled . comparisons of cohort participants with all women giving birth in norway during the same period identified several deviations in prevalence estimates , and of particular interest to the present study is the under - representation of single - mothers , women below 25 years of age , and that infants in moba had a somewhat higher mean birth weight and were on average born after longer gestation . in terms of birth weight and gestational age , the fact that the youngest women are less represented , and as young women has the overall lowest employment rates [ 29 , 30 ] , may imply a bias towards higher work participation in this sample as compared to the general population . as somewhat older women tend to have completed their education , are more likely to be stable employed , and thus to experience a more favorable economic situation , they may also have better opportunity to temporarily stay at home or to reduce work hours . nevertheless , the overall employment rates in the present sample correspond fairly well with that of women with young children in the general norwegian population . moreover , apart from information reflecting single - mother versus two - parent households , the study lacks objective information on paternal factors such as educational attainment and employment status . however , although the total household income is dependent on such factors , and further , that household income is likely to impact on the employment choices of women with small children , the subjective assessment regarding dependency on own income used in this study may better reflect the individual realities as they are perceived by the members of the household as a whole . notwithstanding the limitations above , as informed consent to participate in the study was obtained during early pregnancy , prior to any knowledge of pregnancy outcomes or children s health care needs , and as the register - based data ensures complete follow - up of all participants who were residents of norway in 2010 , the present study constitutes a powerful alternative to traditional longitudinal approaches which often suffer from a large loss to follow - up and systematic attrition . in accordance with other studies on employment among mothers of children with shcn , it appears that mothers of lbw children do modify their employment behavior in order to meet the health care needs of their children . their higher risk of non - employment may result in these women becoming increasingly disadvantaged with regard to future career prospects , earnings potential , and pension benefits . although our sample comprises only young children , many of the conditions prevalent in lbw children persist throughout adolescence and young adulthood , while other conditions have a later onset and are discovered only as the child grows older [ 39 , 40 ] . as lbw children are at higher risk of future academic under - achievement , unemployment , and being granted early disability pension , this implies that the health problems and care needs of many of these children persist for an extended period of time in which other healthy children gradually become more independent . their care needs are thus likely to inhibit future maternal employment prospects also as the children grow older . disrupted occupational careers may prevent many women from utilizing the beneficial effects of stable employment , possibly resulting in poorer caregiver health that in turn can have detrimental effects for the welfare of the entire family in the long run [ 44 , 45 ] . in order to reduce long - term negative consequences of non - employment among those who have a preference to participate in the labor market , it will be important to initiate arrangements for these mothers that make it possible to remain employed while at the same time meeting the care needs of their children .
introduction . numerous devices have been introduced for correction of class iii malocclusion and maxillary deficiency . aim . to assess the dentoskeletal effects of miniplates combined with class iii traction in treating cl iii malocclusion and maxillary deficiency in growing patients . methods . this case describes the treatment of a maxillary - deficient 11-year - old boy by using miniplates . the patient 's parents rejected the use of extraoral appliances and major surgical correction ; therefore the treatment was done by using class iii elastics connected from two mandibular miniplates to an upper removable appliance . two miniplates were inserted in the anterior part of the mandible in the canine areas under local anaesthesia . the treatment lasted for 10 months after which favourable correction of the malocclusion was observed . results . the sna and anb angles increased by 5.1 and 4.4 , respectively . lower 1 to mandibular plane decreased by 3.4. conclusions . this case demonstrates that miniplates can be a suitable method to extraoral appliances and major surgery in maxillary deficiency cases .	skeletal class iii anomalies are associated with maxillary retrusion , mandibular protrusion , or both [ 1 , 2 ] . it has been found that 65% to 67% of all class iii malocclusions were characterized by maxillary deficiency . in subjects with maxillary deficiency where the mandible is not markedly affected various types of extraoral appliances , such as facemasks and reverse pull headgears have been used to correct maxillary deficiency ; [ 46 ] however , there are problems with patient compliance due to their size and appearance . dental implants , miniplates , and modified fixation screws provide bone anchorage in orthodontic treatment [ 79 ] . miniscrews ( mini - implants ) have also become popular because they are easier to both insert and remove [ 10 , 11 ] . in this case report , two miniplates were inserted in the anterior part of the mandible in the canine areas and connected to a removable appliance in the upper jaw by use of elastics in order to correct maxillary deficiency . the patient was an 11-year - old boy who was referred for treatment of maxillary deficiency . he had no medical problems , and there were no signs of temporomandibular joint dysfunction . the facial photographs showed a class iii appearance with a concave profile because of maxillary deficiency . the pretreatment intraoral photographs and dental casts showed class iii relationship of the central incisors and anterior crossbite . the patient had a class iii molar relationship on the right and class i on the left side ( figures 1 and 2 ) . cephalometric analysis confirmed the class iii skeletal pattern ( table 1 ) ( figure 3 ) . the treatment objectives for this patient were to correct the deficient maxillary arch , ideally by forward positioning of the maxilla ; obtain an ideal overjet and overbite ; correct the anterior crossbites . extraoral appliances , such as protraction facemask , class iii functional appliance , any modified maxillary protraction devices , and orthognathic surgery , were considered as alternative treatments for the correction of this class iii malocclusion . however , the patient refused the use of extraoral appliances and major surgery . therefore , in this case , it was decided to use miniplates to protract the maxilla by application of class iii elastics . plates for orthodontic anchorage ( junji sugawara , d.d.s . , ph.d . ) ( ap - yl-013 ) were placed under local anaesthesia in the canine areas of the mandible by a maxillofacial surgeon . the ideal position for miniplates insertion was evaluated by using a panoramic radiograph in order to avoid damage to the roots of the adjacent teeth and mental foramen . a tightly fitting and well - retained upper removable appliance was fabricated with two adams clasps on the upper first permanent molars . each of the adams clasps had a loop which was used for retaining the elastics . a maxillary posterior bite plate was used to disclude the upper and lower jaws . unitek elastics ) were connected from the hooks of the miniplates to the adams clasps of the removable appliance to generate approximately 500 g of anterior retraction . the patient was instructed to wear the appliance full - time except for eating , contact sports , and tooth brushing ; he was also told to change the elastics every day . in order to retain these elastics , the adams clasps on the molars were bent to form loops ( figure 4 ) . after 10 months of active treatment a positive overjet and class i buccal segments were achieved and the anterior crossbite was corrected ( figures 5 and 6 ) . the posttreatment cephalometric radiograph tracing showed a favourable increase of 5.1 and 4.4 in the sna and anb angles , respectively , ( figure 7 ) . the pre- and posttreatment cephalometric superimposition on the anterior cranial base is shown in figure 8 . this case demonstrates the clinical application of miniplates in the treatment of an 11-year - old boy with maxillary deficiency . our system of treatment differs from conventional force applications , such as facemasks [ 46 ] . previous studies [ 2 , 1220 ] show that a significant amount of maxillary forward movement can be produced with maxillary protraction appliances . recent reports indicate that some anteroposterior changes can be achieved up to the beginning of adolescence ; however , these appliances may cause great discomfort for patients and are highly visible to wear , which leads to reduced patient cooperation . another problem caused by extraoral appliances is that they can cause skin abrasions on the chin especially in hot climates . therefore patients may simply refrain from wearing the appliance , and the lack of cooperation might lead to an unsatisfactory result . one of the disadvantages of extraoral appliances is that , when extraoral force is applied against the chin , it is difficult to avoid tipping the lower incisors lingually . in other words , use of a chin cup can lead to lingual tipping of the lower incisors as a result of the pressure of the chin cup component on the lower lip and dentition . in most cases , lingual tipping is an undesirable side effect and can cause crowding . in a case report miniscrews have been used for treatment of maxillary deficiency . one of the limitations of miniscrew is their loosening , which can be distressing for the clinician and the patient . in order to overcome this problem de clerck et al . used the miniplates to protract the maxilla however ; the design of current case report is different from that study . in a recent study bone - anchored maxillary protraction ( bamp ) with miniplates was used in patients with class iii malocclusion , and significant improvements of over jet and molar relationship were recorded . in this case report , minor surgery and miniplates were used to overcome these various problems . as undertaken in this case , applying a force to the teeth in order to correct the skeletal discrepancy will inevitably result in tooth movement ; therefore , a full coverage upper removable appliance was used to cover all the maxillary dentition . however , since the patient was only 11 years old and still had considerable residual growth , treatment was continued by fixed appliance . one force component is in a horizontal direction , moving the maxilla forwards , which is favourable in maxillary deficiency cases . the second component is in a vertical direction , moving the posterior maxillary dentition downwards . this might lead to unfavourable tooth movements in high angle cases , but it is not a problem in patients with a low or average face height . maxillary posterior bite plate can overcome this problem in high angle cases by decreasing facial height . this case report demonstrates a different method of using miniplates to treat an 11-year - old boy with a skeletal class iii malocclusion and maxillary deficiency . this treatment was found to be an acceptable alternative to the use of extraoral appliances such as facemasks and major surgery .
backgroundalthough a myocardial bridge ( mb ) is often regarded as a benign coronary variant , recent studies have associated mb with focal myocardial ischemia . the physiological consequences of mb on ventricular function during stress have not been well established.methods and resultswe enrolled 58 patients with mb of the left anterior descending artery , diagnosed by intravascular ultrasound . patients underwent invasive physiological evaluation of the mb by diastolic fractional flow reserve during dobutamine challenge and exercise echocardiography . septal and lateral longitudinal strain ( ls ) were assessed at rest and immediately after exercise and compared with strain of matched controls . absolute and relative changes in strain were also calculated . the mean age was 42.516.0 years . fiftyfive patients had a diastolic fractional flow reserve 0.76 . at rest , there was no significant difference between the 2 groups in septal ls ( 19.01.8% for patients with mb versus 19.21.5% for control , p=0.53 ) and lateral ls ( 20.12.0% versus 20.01.6% , p=0.83 ) . with stress , compared with controls , patients with mb had a lower peak septal ls ( 18.92.6% versus 21.71.6% , p<0.001 ) and lower absolute ( 0.12.1% versus 2.51.3% , p<0.001 ) and relative change ( 0.611.2% versus 13.17.8% , p<0.001 ) in septal ls , whereas there was no significant difference in lateral ls . in multivariate analysis , diastolic fractional flow reserve and length were independent determinants of lower changes in septal ls.conclusionspatients with a hemodynamically significant mb , determined by invasive diastolic fractional flow reserve , have significantly lower change in septal ls on exercise echocardiography , suggesting that septal ls may be useful for noninvasively assessing the hemodynamic significance of an mb .	a myocardial bridge ( mb ) is a common coronary variant that occurs when one of the coronary arteries tunnels through the myocardium rather than resting on the epicardium . the prevalence of mb varies depending on the cohort studied , but invasive studies with intravascular ultrasound ( ivus ) and computed tomographic angiography suggest a prevalence of 25%.1 , 2 although often regarded as benign , mbs have been associated with ischemia,3 acute coronary syndrome involving the proximal left anterior descending artery,4 coronary spasm,5 arrhythmia including both supraventricular and ventricular tachycardia,6 potentially early death after cardiac transplantation,7 and sudden cardiac death.8 identifying an mb among patients with chest pain syndrome is challenging . the current gold standard for detecting an mb is ivus , with much greater sensitivity than coronary angiogram by both systolic compression3 and a characteristic echolucent halfmoon sign within the mb.9 more recently , computed tomographic angiography has been shown to be valuable in diagnosing the presence of an mb.10 functionally , escaned et al highlighted the importance of diastolic fractional flow reserve ( dffr ) during dobutamine challenge , as opposed to adenosine challenge and measurement of a mean fractional flow reserve , to assess the hemodynamic severity of an mb.11 in addition , abnormal septal motion and myocardial strain have been increasingly associated with the presence of an mb . our group previously described a distinctive septal wall motion abnormality with apical sparing in patients with an mb.12 jhi et al reported that reduced left ventricular ( lv ) strain has been identified during dobutamine challenge in patients with an mb,13 and wang et al reported a decrease in resting strain in the mb perfusion territory.14 for this study , we hypothesized that patients with an mb would have a reduced septal longitudinal strain ( ls ) and comparable lateral ls during exercise compared with age and sexmatched controls . we further hypothesized that the degree of impairment would be associated with the hemodynamic severity of the mb based on dffr . we identified 65 patients with an mb in the left anterior descending artery , diagnosed by ivus , who had undergone an evaluation of dffr during dobutamine challenge and an exercise echocardiogram at our institution . seven patients were excluded because they had previous cardiac surgery or a significant coronary artery stenosis ( > 50% ) by coronary angiography . the patients had been referred for invasive testing for refractory unexplained exertional chest pain with noninvasive imaging suggestive of an mb ( septal wall buckling with apical sparing on stress echocardiography).12 the patients with an mb were compared with 50 age and sex matched controls who were also matched for work load and peak blood pressure during exercise . controls had undergone stress echocardiography and had no history or echocardiographic findings of mb , coronary artery disease , valvular heart disease , lv dysfunction , or lv hypertrophy . all study participants reached target heart rate ( 85% of maximum predicted heart rate ) during exercise . this study was approved by stanford university 's institutional review board , and informed written consent was obtained from all patients . ivus image acquisition was performed with a 40mhz mechanical transducer ultrasound catheter ( atlantis sr pro2 or opticross ; boston scientific , marlborough , massachusetts ) in the left anterior descending artery with placement of the ivus sensor as far distally as safely possible . the presence of an mb was defined by the identification of an echolucent halfmoon sign on ivus . mb length was measured by the distance from the first proximal appearance of the echolucent halfmoon sign to its distal end . combowire recordings were stored on the combomap console ( volcano , san diego , california ) for offline analysis . all patients then underwent hemodynamic testing of the mb using the invasive dobutamine challenge protocol described below . hemodynamic measurements were made using the combowire xt pressure and flow wire ( volcano ) . pressure and flow velocity waveforms were recorded proximal to , within , and at least 1 cm distal to the mb , identified by ivus , at baseline and peak dobutamine stress . dobutamine was given intravenously in increments of 10 to 20 g kg min every 3 minutes ( a maximal dose of 50 g kg min with up to 1.0 mg atropine ) until the maximum predicted heart rate on previous exercise echocardiography was achieved . using a digital electronic caliper , instantaneous dffr ( the fraction of diastolic coronary artery pressure divided by diastolic aortic pressure ) at rest and with stress was calculated proximal to , within , and distal to the mb , as described previously12 ( figure 1 ) . a dffr 0.76 was considered to be hemodynamically significant in this study , as in the previous study by escaned et al.11 pressure curves of a representative case of patients with a myocardial bridge ( mb ) . the red curve shows the aortic pressure , and the yellow curve shows the coronary pressure within the mb . both pressures are similar at baseline ; however , coronary systolic pressure increases with stress , whereas diastolic pressure decreases compared with aortic pressure . all echocardiographic studies were performed using commercially available echo systems ( sonos 7500 , ie33 , and epiq 7c ; philips medical imaging , eindhoven , the netherlands ) . standard echocardiographic views , including parasternal long and shortaxis and apical 2 , 3 , and 4chamber views , were obtained in 2dimensional and color tissue doppler modes . lv ejection fraction was derived from the modified simpson method according to the american society of echocardiography ( ase ) guidelines.15 exercise echocardiography was performed according to the ase recommendations.16 an average of 5 to 10 beats per loop was recorded at rest and immediately after stress . all participants performed the bruce treadmill protocol test17 with a target heart rate of 85% of maximum predicted for age . lv ls was assessed from the 4chamber view both at rest and with exercise , and the absolute change in strain ( exercise ls minus rest ls ) and the relative change in strain ( exercise ls minus rest ls divided by rest ls ) were evaluated . strain values were obtained by manual tracing on an offline xcelera workstation ( philips medical imaging ) , for which the frame rate was 50 to 52 hz to ensure the best measurement quality ; postprocessing analysis of peak exercise does not always yield optimal tracking . initial length was obtained in enddiastole ( peak of qrs ) , and final length was obtained in endsystole ( the smallest lv volume ) . to assess more detailed lv strain , septal and lateral ls and global ls ( gls ) were assessed , as shown in figure 2 . for quality control , the same mitral annular reference points were used ; in addition , the apical reference point was determined to be the furthest point of the left ventricle from the mitral annulus , and it was kept stable to avoid overestimating strain measures due to foreshortening . foreshortening was defined as being when the endocardial border of the apex was significantly displaced in systole . in fact , magnetic resonance based studies have shown that the endocardial border of the apex usually displaces about 1.5 mm in systole.18 one representative beat with minimal respiratory or translational motion was chosen to trace in both enddiastole and endsystole . all strain values were calculated by the following formula in which l0 indicates initial length and l1 indicates final length : strain ( % ) = 100(l1l0)/l0.19 prior to performing the manual tracing , we compared strain values derived from manual tracing and the software analysis ( tomtec imaging system , unterschleissheim , germany ) in 50 healthy persons and found a relative difference 4.84.3% . a more negative value represents better function . for the presentation of strain values in this study , initial length ( l0 ) was obtained in enddiastole , and final length ( l1 ) was obtained in endsystole . to divide left ventricle longitudinal components into septal and lateral segments , the apex was standardized as midventricle from the middle of the transverse segment in both systole and diastole . strain ( % ) = 100(l1l0)/l0 . variables are presented as count and percentage or mean and standard deviation ( sd ) for normally distributed variables . variables were compared with ( student or welch ) t test or mann whitney u test , as appropriate . multivariate regression analysis was performed to identify the independent correlates of absolute and relative changes in strain . we used a regression model that included age , sex , mb length , systolic blood pressure , peak heart rate , and dffr either within or distal to the mb . comparisons of strain values between baseline and stress were analyzed by paired t test . for inter and intraobserver variability , 15 patients with an mb and 15 controls were randomly selected and septal ls , lateral ls , and gls were blindly reanalyzed 2 to 4 weeks after the first analysis without references to the initial tracings , and the absolute difference was assessed . all analyses were performed using spss 21 software ( ibm corp , armonk , ny ) . table 1 shows the clinical and echocardiographic characteristics of the age , sex , and blood pressure matched patients with an mb and controls . patient characteristics bp indicates blood pressure ; bpm , beats per minutes ; bsa , body surface area ; ivsd , diastolic interventricular septum ; la , left atrial ; lv , left ventricular ; lvef , left ventricular ejection fraction ; mb , myocardial bridge ; pwd , diastolic posterior wall ; svi , stroke volume index . coronary angiography found that no patient with an mb had significant epicardial coronary disease ( 50% stenosis ) . men had longer mbs than women ( 32.613.9 versus 23.112.8 mm , respectively ; p=0.02 ) . the dffr was 0.76 in 55 patients either within or distal to the mb , whereas it was > 0.76 in 3 patients at both sites . because the number of patients with a dffr > 0.76 was extremely small , further analysis was performed only in patients with a dffr 0.76 . assessment of hemodynamics both within and distal to an mb was possible in 40 patients ; 13 patients had a dffr 0.76 only within the mb , 4 had a dffr 0.76 only distal to the mb , and 23 had a dffr 0.76 both within and distal to the mb ( figure 3 ) . of the rest , 14 patients had a dffr 0.76 within the mb , but no data were obtained distal to the mb because the length of the mb precluded safe placement of the combowire in the distal vessel . one patient had a dffr 0.76 distal to the mb , but no data were obtained within the mb because the length of the mb was too short to accurately measure dffr within it . mbs were significantly longer in patients whose distal dffr was 0.76 compared with patients whose distal dffr was > 0.76 ( 25.613.0 versus 17.98.1 mm ; p=0.03 ) . some patients with an mb showed full recovery of dffr distal to the mb ( a ) . indicates dffr > 0.76 ; + , dffr 0.76 ; dffr , diastolic fractional flow reserve ; mb , myocardial bridge . in terms of stress lv segmental wall motion was normal in all patients , and lv ejection fraction and stroke volume index at rest were not significantly different between the 2 groups . with exercise , abnormal septal motion was found in 50 patients with an mb , whereas no wall motion abnormality was found in controls . lv ejection fraction and stroke volume index were significantly lower in patients with an mb compared with controls . in the mb group , 33 patients ( 60% ) exhibited chest pain , 13 ( 24% ) presented with stsegment depression ( defined as stsegment depression 1 mm ) on ecg , and one had a 3beat run of ventricular tachycardia during exercise . in the control group , nobody presented with stsegment depression on ecg or chest pain ( table 1 ) . regarding strain assessment , at baseline , there was no significant difference between the 2 groups for all strain values : septal ls was 19.01.8% for patients with an mb versus 19.21.5% for controls ( p=0.58 ) ; lateral ls was 20.12.0% versus 20.01.6% ( p=0.53 ) , respectively ; and gls was 19.51.8% versus 19.71.4% ( p=0.68 ) , respectively . with exercise , in the mb groups , septal ls did not change significantly ( from 19.01.8% to 18.92.6% ; p=0.83 ) , whereas lateral ls increased significantly ( from 20.12.0% to 22.82.9% ; p<0.001 ) . in contrast , in the control group , both septal and lateral ls increased significantly ( from 19.21.5% to 21.71.6% and from 20.01.6% to 22.71.8% ; p<0.001 for both ) ( figure 4a and 4c ) . the absolute and relative changes in septal ls were significantly smaller in patients with an mb than in controls ( 0.12.1% versus 2.51.3% for absolute change , and 0.611.2% versus 13.17.8% for relative change ; p<0.001 for both ) . in contrast , the absolute and relative changes in lateral ls were similar between the 2 groups ( p=0.97 and p=0.98 , respectively ) ( figure 4b and 4d ) . , there was a significant difference between the 2 groups in strain with stress ( a ) and in absolute and relative changes ( b ) . in contrast , in lateral segments , there was no significant difference in strain with stress ( c ) and absolute and relative changes ( d ) . the absolute and relative changes in septal ls had a better positive correlation with the dffr distal to the mb ( r=0.52 , p<0.001 for septal absolute change and r=0.52 , p=0.001 for septal relative change ) than the dffr within the mb ( table 2 ) . the absolute and relative changes in septal ls also had a moderate negative correlation with the length of the mb ( r=0.32 , p=0.02 for absolute change and r=0.32 , p=0.02 for relative change ) . in multivariate analysis , a lower dffr distal to the mb and male sex were independent correlates of lower absolute and relative changes in septal ls . when the dffr within the mb was included instead of the dffr distal to the mb , a lower dffr within the mb and male sex were independent correlates of a lower absolute change in septal ls ; however , male sex was the only independent correlate of a lower relative change in septal ls ( table 2 ) . although the length of the mb was not left in the multivariate analysis when included as a continuous variable , it was left as an independent correlate of absolute change in septal ls instead of male sex when it was categorized using the median value . correlation between absolute and relative changes in septal ls and dffr values pattern 1 includes dffr distal to the mb . dffr indicates diastolic fractional flow reserve ; ls , longitudinal strain ; mb , myocardial bridge . mb length was left as an independent correlate of absolute change in septal ls instead of male sex when it was categorized using the median value . in terms of the inter and intraobserver variability of this study , the absolute differences between the 2 investigators were 1.91.4% for septal ls , 2.21.2% for lateral ls , and 1.91.0% for gls at rest and 2.61.6% for septal ls , 2.81.9% for lateral ls , and 2.21.6% for gls immediately after stress . the absolute differences within the observer were 1.61.1% for septal ls , 1.40.7% for lateral ls , and 1.10.8% for gls at rest and 1.71.1% for septal ls , 1.51.1% for lateral ls , and 1.20.9% for gls immediately after stress . the main finding of our study is that patients with hemodynamically significant mbs have no increase or have a blunted increase in septal ls with exercise compared with controls . moreover , the lack of significant increase in septal ls appears to be associated with the length of the mb and the hemodynamic significance assessed by dffr . these functional characteristics may be helpful in the assessment of patients referred for unexplained exertional chest pain . fractional flow reserve is the invasive reference standard to assess the hemodynamic significance of a fixed coronary artery stenosis . in a recent study , johnson et al demonstrated that fractional flow reserve during adenosine infusion is an independent and strong correlate of outcome : patients with lower fractional flow reserve have a higher likelihood of adverse events.20 assessing the hemodynamic significance of an mb , however , may be more challenging . in patients with an mb , dffr may be more reliable than conventional mean fractional flow reserve to assess hemodynamic significance because the increased systolic pressure during compression and the decreased diastolic pressure may offset each other ( figure 2).11 in addition , the use of dobutamine rather than adenosine has been shown to be important in evaluating an mb . adenosine results in maximal hyperemia through vasodilation of microcirculation , which is effective for a fixed stenosis , but the chronotropic and inotropic effects of dobutamine are necessary to evaluate the hemodynamic significance of the dynamic stenosis caused by an mb.21 although the cutoff value to determine hemodynamically significance has been argued , a dffr 0.76 was considered to be hemodynamically significant in this study , as in the previous study by escaned et al.11 from a pathophysiological perspective , lin et al previously proposed a model of ischemia associated with an mb involving the venturi effect.12 lumen area within the mb decreases during endsystole to early diastole ( figure 5a ) , and the compression resembles the narrowed section of a pipe . pipe , the fluid velocity must increase ( figure 5b ) to satisfy the principle of conservation of energy by the continuity equation . at the same time , the pressure must decrease to satisfy the principle of the bernoulli equation , thus the pressure is decreased in the tunneled section . with this model , pressure should recover distally , provided the diameter of the distal arterial segment is larger than in the tunneled segment.22 proposed schematic of ischemia from mb . a , normal vessels show the pattern in which an increase in area is seen throughout systole with the rise in aortic pressure ( solid line ) . in contrast , mb segments undergo a marked decrease in area , particularly in the second half of systole . importantly , this vessel compression persists into early diastole ( dotted line).3 b , venturi effect . when fluid is passing the narrow lesion , its velocity increases by the principle of energy conservation , and pressure decreases by the principle of the bernoulli equation . distally , the velocity decreases and the pressure increases to recover by the same principles . hemodynamic significance of an mb has been assessed invasively , and few studies have evaluated noninvasively . the key findings of our study are that change in strain with stress may be useful in identifying the hemodynamic significance of an mb and that the more hemodynamically significant it is ( the lower the dffr ) , the more impaired is the change in septal ls with stress . in our study , change in septal ls showed a stronger correlation with the dffr distal to the mb than with the dffr within the mb . most of our patients with a significant dffr distally also had a significant dffr within the bridge , and we suspect that mbs with a significant dffr distally may be exposing an even more extensive area of myocardium to ischemia . the primary reason for this phenomenon may be a longer mb delaying the pressure recovery , as shown in the relationship between septal ls and mb length . likewise , male sex was an independent correlate of a diminished increase in septal strain with stress . a possible explanation may be the interrelationship between male sex and the length of mb , as suggested in the multivariate analysis . along with the possible reasons for preventing full recovery of the pressure distally may be a small caliber vessel beyond the bridge , a short segment of vessel beyond the bridge , inability of the combowire to be placed far enough distally to detect recovery , or the undetected presence of further mb distal to the investigated area . al recently published a study demonstrating resting ls of the left anterior descending artery territory by 3dimensional echocardiography being decreased.14 in contrast , we did not find that ls at rest was useful in identifying hemodynamically significant mbs , but it may be because 3 dimensions are more able to detect subtle changes in strain than 2 dimensions . our study also consisted of a younger population and exposed patients to a shorter duration of ischemia compared with the population in the study by wang et al . clinically , our study may be of value in the assessment of patients with exertional chest pain of undetermined etiology . if an mb is detected by exercise echocardiography ( by the presence of focal septal buckling with apical sparing ) and/or confirmed by multisliced computed tomography or ivus during coronary angiography , determination of impaired strain increase on exercise echocardiography may suggest that the mb is hemodynamically significant . furthermore , if the distinctive septal wall motion pattern on exercise echocardiography is proven to be accurate enough for the diagnosis of an mb,12 change in strain may be useful in stratifying the patients with an mb according to hemodynamic significance , possibly offering a totally noninvasive diagnosis of mb by exercise echocardiography . another consideration to enhance image quality and feasibility of speckle tracking is to perform dobutamine stress echocardiography , although further validation study is needed . first , the number of patients enrolled was small ; however , the study is the largest of patients with an mb who underwent both invasive physiological evaluation and exercise echocardiography . second , the strain values were obtained by manual tracings because of the difficulty in obtaining reliable speckle tracking images during exercise ; however , we showed good inter and intraobserver variability , even for stressbased images . third , because we can not yet provide longterm followup data for our cohort , we can not determine how change in septal strain is useful in predicting cardiovascular outcomes in patients with an mb . fourth , all patients with an mb evaluated in this study had a dffr 0.76 . further study is needed to assess the change in septal ls during stress in patients with an mb who have a dffr > 0.76 . moreover , a dffr threshold of 0.76 was used in this study , as in previous studies11 ; however , the cutoff value has not been well discussed , and whether or not a dffr 0.76 truly represents hemodynamic significance is unknown . in addition , we still can not say with certainty whether the strain pattern is due to ischemia or to an anatomic variation from having a tethered portion of left anterior descending artery . first , the number of patients enrolled was small ; however , the study is the largest of patients with an mb who underwent both invasive physiological evaluation and exercise echocardiography . second , the strain values were obtained by manual tracings because of the difficulty in obtaining reliable speckle tracking images during exercise ; however , we showed good inter and intraobserver variability , even for stressbased images . third , because we can not yet provide longterm followup data for our cohort , we can not determine how change in septal strain is useful in predicting cardiovascular outcomes in patients with an mb . fourth , all patients with an mb evaluated in this study had a dffr 0.76 . further study is needed to assess the change in septal ls during stress in patients with an mb who have a dffr > 0.76 . moreover , a dffr threshold of 0.76 was used in this study , as in previous studies11 ; however , the cutoff value has not been well discussed , and whether or not a dffr 0.76 truly represents hemodynamic significance is unknown . in addition , we still can not say with certainty whether the strain pattern is due to ischemia or to an anatomic variation from having a tethered portion of left anterior descending artery . patients with a hemodynamically significant mb have a reduced change in septal strain on exercise echocardiography . the degree of reduction is associated with the severity of hemodynamic significance , as determined by invasive dffr . assessment of change in septal strain may be clinically useful in the noninvasive evaluation of patients with chest pain .
an estimated 10 billion dollars is spent treating gastro - oesophageal reflux disease ( gerd ) in the usa every year . the present article reports a case of the safe and successful use of transoral incisionless fundoplication ( tif ) using the esophyx90 device in the surgical treatment of gerd .	gerd is usually characterized by symptoms caused by esophageal mucosal exposure to acidic gastric contents . the pathophysiology is related to failure of the lower esophageal sphincter ( les ) to act as a barrier to gastric reflux . the adequacy of les function is dependent on its pressure , total length and portion exposed to positive intra - abdominal pressure . defects in one or a combination of the above factors will lead to reflux . ( 1 ) the prevalence of gerd has been increasing for the last 2 decades . ( 2 ) it affects 10 - 20 % of the adult population in the developed world , with its highest prevalence in north america . proton pump inhibitors ( ppis ) have been shown by multiple randomized controlled trials ( rct ) to be the most effective medical therapy for gerd . several large meta - analysis of multiple rct showed that ppis are superior to histamine 2 receptor blockers ( h2rb ) in short term treatment of esophagitis and long term maintenance therapy to prevent symptomatic relapse and endoscopicaly proven esophagitis . , surgery should be considered in patients with progressive disease , extraesophageal symptoms , and gerd - related complications such as strictures or barrett 's esophagus . it should also be considered in younger patients intolerant of ppis or post menopausal female patients at risk for fractures due to ppi - induced calcium malabsorption . laparoscopic nissen fundoplication , which involves a 360 degree wrap of the gastric fundus around the esophagus at the gastro - esophageal junction , is the current surgical procedure of choice for severe , refractory gerd . studies have shown over 90 % success in symptom resolution , improved quality of life and overall improvement of general health in over 70 % of patients . reoperation for symptoms of heartburn , dysphagia , bloating and inability to belch due to disruption or slippage of a fundoplication has been reported in up to 7% of patients after laparoscopic nissen fundoplication . the patient was a twenty nine years old male with long standing gastro - esophageal reflux disease ( gerd ) who had been treated with high dose proton pump inhibitors ( ppis ) for over 3 years with little response . the patient complained of symptoms of heartburn gradually progressing to throat pain , ear pain and tinnitus . symptoms would worsen when laying supine , after a large meal and with specific foods such as chocolate , spicy foods and alcohol . the patient had some relief of his symptoms with ppis initially but then the symptoms recurred . pre - operative upper endoscopy showed a hill grade i gastro - esophageal valve , ( fig . motility study showed only 25% of swallows produced peristaltic wave throughout the esophagus with a mean distal amplitude of 77mmhg . the remaining swallows produced a mixture of low amplitude synchronous waves , partially propagated and non - propagated waves . the lower esophageal sphincter ( les ) measured to 3 cm , with 1 cm above the diaphragm . the les had a mean resting pressure of 9 mmhg , which relaxed with swallowing . a twenty four hour ph study produced a demeester score of 20.25 ( normal range below 14.5 ) . pre - operative egd showing hill grade i gastro - esophageal valve the patient elected to undergo a transoral incision - less fundaplication , in which a 4 cm gastro - esophageal valve was created after the endoscopic application of 14 polypropylene transmural fasteners through the gastric fundus and the distal esophagus ( 1 - 3 cm above the z - line ) . the procedure also produced a 270 degree endoluminal wrap of the fundus around the intra - abdominal esophagus . there were no major intra - operative complications , minimal intra - operative blood loss and the patient was discharged home the morning after surgery . two months after the procedure , the patient 's symptoms of reflux had significantly resolved . he was almost completely off his ppis , he could eat many of the foods which gave him reflux before surgery without any difficulty and he could now sleep lying flat , something which he had been unable to do for three years . a novel approach in the evolution of the surgical treatment of gerd is the transoral incisionless fundoplication ( tif ) . tif employs a tissue plication device called esophyx ( endogastric solutions , redmond , wa , usa ) that endoscopically replicates a 270 - 320 degree gastric fundoplication to create a 3 - 4 cm neo - valve at the ge junction . the procedure is done under general anesthesia , with either oro - tracheal or naso - tracheal intubation , with the patient positioned in either the left lateral decubitus or supine position on the operating table . the operating team consists of an endoscopist who provides continued retroflex visualization of the ge junction and a second operator , usually a surgeon , who performs the actual tissue manipulation and plication . after a single transoral introduction of the instrument over the endoscope , this creates a 3 - 4 cm long sleeve of tissue that is plicated by 6 mm h - shaped polypropylene fasteners in a 200 to 300 degree fashion . full thickness serosa - serosa plication at 1 - 2 cm above the gastro - esophageal junction begins with deployment of fasteners at the posterior and anterior side as visualized with the endoscope in the retroflexed view . plication then continues at the greater curvature , 3 - 4 cm above the gastro - esophageal junction as the tissue is being pulled down . ( fig 3 ) by the end of the procedure ( which typically takes 45 - 75 minutes ) , tif increases the length of intra - abdominal esophagus , reduces small hiatal hernias , creates a 3 - 4 cm ge valve and recreates the angle of his . full thickness serosa - serosa plication at 1 - 2 cm and 3 - 4 above the gastro - esophageal junction . the performance of tif with esophyx has been fda approved in the usa since september 2007 . over the last 2 and half years , 2000 cases have been performed in the usa . cadiere and his group were able to show no adverse effects , greater then 50% improvement in gerd - hrql scores , elimination of heartburn in 93% of patients , ppi use in 71% of patients and esophagitis in 55% of patients at 2 year follow up . ( 6 ) newer unpublished data from two case series ( n = nearly 70 patients ) from two independent investigators in united states demonstrate normalization of esophageal ph in 76 % of patients and complete independence from ppi 's in approximately 90% of patients . 87% of subjects were satisfied and 82% were asymptomatic based on hrql and gsrs scores . tif with esophyx is effective in decreasing symptom severity , daily ppi requirements and improving quality of life ( in the short term ) in patients with moderate to severe gerd . tif with esophyx successfully increases les resting pressure , decreases acid reflux and supports esophageal healing in chronic gerd . long term studies are needed to establish whether it is will be as durable as more traditional forms of anti - reflux surgery .
large bowel obstruction ( lbo ) is most commonly due to malignancy , volvulus , hernia , diverticular disease and inflammatory bowel disease . lbo due to adhesions is unusual . a literature review was conducted which revealed that only a few such cases have been reported . we report two cases of lbo secondary to adhesions in patients , one with and one without a past abdominal surgical history . we highlight that while rare , the aetiology of lbo secondary to adhesions must be considered in the differential diagnosis in patients presenting with obstructive symptoms .	the most common causes of large bowel obstruction ( lbo ) are colonic neoplasm , volvulus , hernia , diverticular disease and inammatory bowel disease . adhesions secondary to previous inammation or operation commonly cause small bowel obstruction , but are a rare cause of lbo . a 58-year - old man presented with sudden onset of right - sided colicky abdominal pain associated with nausea and vomiting . he had three similar episodes of pain during the previous 3 weeks with two hospital admissions . the last admission was 4 days before , and he was in the hospital for 3 days and discharged home after he opened his bowel . his past medical history ( pmh ) included gall stone pancreatitis 10 years ago and laparoscopic cholecystectomy subsequently . on examination , he was afebrile and haemodynamically stable . the abdomen showed fullness with mild tenderness in the right iliac fossa ( rif ) . haemoglobin was 14.9 g / dl and the white cell count ( wcc ) was 9 10/l . other remarkable laboratory tests included a c - reactive protein ( crp ) of 15 mg / l . 1 ) revealed dilatation of a focal segment of large bowel with mildly dilated small bowel . computed tomography ( ct scan ) of his abdomen reported that the gas - filled viscus is likely to represent the caecum ( fig . 2 ) . figure 1:plain axr ( case 1 ) revealed dilatation of a focal segment of a large bowel with a mildly dilated small bowel . figure 2:ct of the abdomen ( case 1 ) demonstrating the gas - filled caecum . plain axr ( case 1 ) revealed dilatation of a focal segment of a large bowel with a mildly dilated small bowel . a presumptive diagnosis of caecal volvulus was made and the patient was taken to theatre . laparotomy revealed an omental band wrapping the colon at the junction between the caecum and the ascending colon with a marked dilatation of the caecum . appendicectomy was performed and the gas in the caecum was aspirated via the appendicular stump prior to its ligation . a 46-year - old woman presented with a 5-day history of absolute constipation , abdominal distension , discomfort and an episode of vomiting . there was no change in her bowel habit and she had no other systemic symptoms . she had a pmh of a right - sided ovarian cyst detected on an ultrasound that she had for lower abdominal pain sometimes ago . a ct scan of the abdomen revealed that the rectum , sigmoid and descending colon were collapsed with an apparent calibre change within the transverse colon with no mass lesion ( fig . figure 3:ct of the abdomen ( case 2 ) showing a calibre change within the transverse colon with no mass lesion . ct of the abdomen ( case 2 ) showing a calibre change within the transverse colon with no mass lesion . a gastrografin enema ( fig . a presumptive diagnosis of transverse colon obstruction was made and the patient was taken to theatre . figure 4:gastrografin enema ( case 2 ) demonstrating no flow beyond the mid - transverse colon . gastrografin enema ( case 2 ) demonstrating no flow beyond the mid - transverse colon . laparotomy revealed an adhesive band extending from the gastrocolic omentum to the right fallopian tube causing a closed - loop obstruction of right half of transverse colon , ascending colon and terminal 5 cm of ileum . appendicectomy was performed and most of the gas in the colon was aspirated via the appendicular stump prior to its ligation . a 58-year - old man presented with sudden onset of right - sided colicky abdominal pain associated with nausea and vomiting . he had three similar episodes of pain during the previous 3 weeks with two hospital admissions . the last admission was 4 days before , and he was in the hospital for 3 days and discharged home after he opened his bowel . his past medical history ( pmh ) included gall stone pancreatitis 10 years ago and laparoscopic cholecystectomy subsequently . on examination , he was afebrile and haemodynamically stable . the abdomen showed fullness with mild tenderness in the right iliac fossa ( rif ) . haemoglobin was 14.9 g / dl and the white cell count ( wcc ) was 9 10/l . other remarkable laboratory tests included a c - reactive protein ( crp ) of 15 mg / l . 1 ) revealed dilatation of a focal segment of large bowel with mildly dilated small bowel . computed tomography ( ct scan ) of his abdomen reported that the gas - filled viscus is likely to represent the caecum ( fig . 2 ) . figure 1:plain axr ( case 1 ) revealed dilatation of a focal segment of a large bowel with a mildly dilated small bowel . figure 2:ct of the abdomen ( case 1 ) demonstrating the gas - filled caecum . plain axr ( case 1 ) revealed dilatation of a focal segment of a large bowel with a mildly dilated small bowel . a presumptive diagnosis of caecal volvulus was made and the patient was taken to theatre . laparotomy revealed an omental band wrapping the colon at the junction between the caecum and the ascending colon with a marked dilatation of the caecum . appendicectomy was performed and the gas in the caecum was aspirated via the appendicular stump prior to its ligation . a 46-year - old woman presented with a 5-day history of absolute constipation , abdominal distension , discomfort and an episode of vomiting . there was no change in her bowel habit and she had no other systemic symptoms . she had a pmh of a right - sided ovarian cyst detected on an ultrasound that she had for lower abdominal pain sometimes ago . a ct scan of the abdomen revealed that the rectum , sigmoid and descending colon were collapsed with an apparent calibre change within the transverse colon with no mass lesion ( fig . figure 3:ct of the abdomen ( case 2 ) showing a calibre change within the transverse colon with no mass lesion . ct of the abdomen ( case 2 ) showing a calibre change within the transverse colon with no mass lesion . a gastrografin enema ( fig . a presumptive diagnosis of transverse colon obstruction was made and the patient was taken to theatre . figure 4:gastrografin enema ( case 2 ) demonstrating no flow beyond the mid - transverse colon . gastrografin enema ( case 2 ) demonstrating no flow beyond the mid - transverse colon . laparotomy revealed an adhesive band extending from the gastrocolic omentum to the right fallopian tube causing a closed - loop obstruction of right half of transverse colon , ascending colon and terminal 5 cm of ileum . appendicectomy was performed and most of the gas in the colon was aspirated via the appendicular stump prior to its ligation . other aetiologies include incarcerated hernias , inflammatory bowel disease , diverticular disease , ischaemic stricture , intussusception and faecal impaction . adhesions , which are brous bands between two or more adjacent anatomic structures , are a common cause of small bowel obstruction but a very rare cause of large bowel obstruction [ 16 ] . the origin of these adhesions can be congenital [ 1 , 2 ] , inammatory , post - traumatic or iatrogenic following previous surgery [ 3 , 4 ] . the aetiology of the bands causing colonic obstruction in our cases was obscure , but may be related to a previous intraperitoneal inflammatory process with gall stone pancreatitis and laparoscopic cholecystectomy in case 1 . interestingly , case 2 had no previous abdominal surgery or any evidence of intraperitoneal inflammatory process . however , unnoticed right - sided salpingitis might have triggered the formation of this band . in the absence of either surgery or abnormal fixation of the colon , adhesive obstruction may be secondary to inflammation of epiploic appendage . in these patients , the inflamed epiploic appendage that has become adherent to the abdominal wall or another intra - abdominal structure constricts the colon directly or forms a band entrapping the small bowel . the sigmoid colon is the most common site for a diseased epiploic appendage , accounting for 50% of patients . other unusual entities causing adhesive colon obstruction include remnants of embryological structures such as vitello - umbilical cord and mesourachus . a vitello - umbilical cord connects the tip of meckel 's diverticulum to the abdominal wall at the umbilicus , obstructing the middle portion of the redundant transverse colon or long sigmoid colon . the mesourachus was bound by adhesions to small bowel mesentery and resulted in severe obstruction of the sigmoid colon , simulating a volvulus . anatomical and histopathological studies of the bands distinguish between embryonic remnants and aberrant mesenteric bands [ 8 , 9 ] . on barium enema , adhesions cause a sharp , localized , circumferential narrowing of the large bowel . occasionally , an area of circular muscular contraction may suggest this appearance . there are some areas of narrowness or focal contraction in the colon that are inconstantly seen radiologically . these contraction rings or colonic valves rarely persist during an entire barium enema , while compression from adhesions does . clinical features and findings of a combination of radiology investigations are important in timely decision - making surgery in patients with an adhesive band causing colonic obstruction . however , it is still difficult to make a preoperative diagnosis of adhesive colonic obstruction .
[ purpose ] an earlier study divided reaching activity into characteristic phases based on hand velocity profiles . by synchronizing muscle activities and the acceleration profile , a phasing approach for reaching movement , based on hand acceleration profiles , was attempted in order to elucidate the roles of individual muscle activities in the different phases of the acceleration profile in reaching movements . [ subjects and methods ] ten healthy volunteer subjects participated in this study . the aim was to electromyographically evaluate muscles around the shoulder , the upper trapezius , the anterior deltoid , the biceps brachii , and the triceps brachii , most of which have been used to evaluate arm motion , as well as the acceleration of the upper limb during simple reaching movement in the reach - to - grasp task . [ results ] analysis showed the kinematic trajectories of the acceleration during a simple biphasic profile of the reaching movement could be divided into four phases : increasing acceleration ( ia ) , decreasing acceleration ( da ) , increasing deceleration ( i d ) , and decreasing deceleration ( dd ) . muscles around the shoulder showed different activity patterns , which were closely associated with these acceleration phases . [ conclusion ] these results suggest the important role of the four phases , derived from the acceleration trajectory , in the elucidation of the muscular mechanisms which regulate and coordinate the muscles around the shoulder in reaching movements .	the ability to reach , grasp , transport , and release objects is essential for performing activities of daily living ( adl)1 . the adl that require large glenohumeral elevation angles like combing hair and reaching are comparable to abduction and forward flexion motions2 . this implies that rotator cuff muscle weakness can also affect the degree of restriction of adl . however , during rehabilitation immediately after surgery , patients may need to learn compensatory movements for adl2 . therefore , reach - to - grasp movement plays an important role in the rehabilitation of patients with shoulder disorders , and interest in the quantitative analysis of upper arm movements is rapidly increasing in biomechanical research and clinical applications . owing to the complexity of the reach - to - grasp movement , grasping movement research still lags behind that of gait . the gait phases can be clearly distinguished : the stride phase , stance phase , and swing phase . in addition , these phases are further divided into sub - phases , which are well established in gait analysis3 . in contrast , the grasping movement is highly complex and depends on many factors , including the position and shape of the target , orientation , perturbation , and presence of obstacles4 . to evaluate the kinematics of the reaching activity , morasso investigated arm trajectories based on hand velocity profiles in 19835 . subsequently , it was proposed that there are two independent components of the grasping movement : the transport component , and the grip component6 . on the basis of this approach , a variety of reports have suggested the presence of a channel controlling hand aperture , which has access to information about the progress of hand transport7 , 8 , indicating the importance of regulation of the transport component . demonstrated that the reach - to - grasp movement can be divided into characteristic phases by simultaneously observing two aspects of the movement , hand transport and finger preshaping , on 3d video recordings . they also suggested the importance of evaluation of reaching movement , that is , hand transport , by quantitative methods from the viewpoint of rehabilitation9 . sabatini10 showed that natural upper - arm movement is composed of sequential phases of reaching , grasping , and retrieval in the horizontal plane , using electromyography ( emg ) and a 3d - motion analysis system . reach - to - grasp movement and hand velocity are regulated rigorously by an integrated complex of multiple muscle activities . since single muscle activity regulates angular velocity in both acceleration and braking , a phasing approach of the reaching movement on the basis of hand acceleration profiles may provide a method for elucidating the role of individual muscles in the reaching movement of the reach - to - grasp task . muscle activities of the upper extremity during reaching movements have been examined by several investigators . sabatini demonstrated that : the upper trapezius activity is tonic when holding the limb up against the force of gravity ; the anterior deltoid activity is spread over the whole duration of the movement ; the biceps brachii activity has a phasic drive at both movement onset and object grasping ; and the triceps brachii activity is essentially tonic10 . prange compared the surface emgs of the muscle activities of healthy elderly people with and without gravity compensation , and demonstrated that gravity compensation only influences the level of muscle activity , not the muscle activation pattern in terms of timing11 . on the basis of these findings , it has been suggested that : the upper trapezius elevates the arm at the start of reaching and retrieval and appropriately positions the scapula during the movements ; the anterior deltoid maintains a certain degree of shoulder abduction , to anteflex the shoulder during reaching and to decelerate retroflexion during retrieval ; the biceps brachii lifts and holds the lower arm above the table and aids in anteflexion of the shoulder ; and the muscle activity of the triceps brachii contributes to extension of the elbow towards the target10 , 11 . it is very important to evaluate reaching with quantitative methods for rehabilitation , in order to objectively assess and describe the coordination and functional status of the impaired upper limb . the analysis of reach - to - grasp phases could potentially help determine how the impairment affects the upper limb movements and to plan the rehabilitation process more effectively . examined the control of movement in the dominant and non - dominant hands , and reported that non - dominant arm motor output was subject to increased variability , which necessitated a greater number of corrective movements12 . the present study focused on the reaching movement , namely , hand transport in the reach - to - grasp task . by synchronizing muscle activities and the acceleration profile , phasing of the reaching movement of the hand based on acceleration profiles , was attempted in order to elucidate the direct role of the individual muscle activities in the different acceleration profiles , differences between the dominant arm and the non - dominant arm muscle activation patterns in the reaching movement . the ultimate goal of the present research is to establish a method of evaluation of hand movements for rehabilitation . ten healthy volunteer subjects ( four males , six females ; age , 21.5 1.0 years ; height , 162.1 8.4 cm ; weight , 55.8 8.6 kg ; mean sd ) with no neurological or muscular disorders , participated in this study . they were all right - hand dominant , and subjects were excluded if they had received surgery related to upper limb structures . the subjects were fully informed about the protocol and gave their informed consent to the experimental procedure , which was approved by the epidemiological research ethics committee of gunma university faculty of medicine ( no . the aim of this study was to evaluate emg and acceleration of the upper limb during a simple reaching movement in a reach - to - grasp task . the task was to move the arm from the initial position in order to grasp a target in front of it . the target - reaching movements were performed in the sagittal plane and corresponded to a cylindrical palmar prehension motion . the measurements were carried out in the order of the dominant and then the non - dominant upper extremity . in the initial position , the measured forearm rested on the table and the shoulder was stabilized in the anatomical reference position with the elbow at 90 flexion . before beginning the movement , the target was placed at a distance of 30 cm in front of the hand on the table . the subjects were informed that no trunk movement was allowed , so they could only move the arm in order to grasp the target . to simulate reaching in normal life , the target was a 0.5 kg cylindrical bottle ( 6.5 cm in diameter ) . at the end of the movement , the average values of the 10 trials were used as representative of each subject , after confirmation of the normal distribution of data using the shapiro - wilk s test . the emg signals of four muscles were recorded , the upper trapezius , the anterior deltoid , the biceps brachii , and the triceps brachii , most of which have been used to evaluate arm motion14 . the subjects were prepared for the placement of emg electrodes by shaving the skin of each electrode site and cleaning it carefully with an alcohol wipe . pairs of ag - agcl pregelled surface electrodes ( dl-141 , s & me co. , japan ) were applied along the muscle fibers over the bellies of the four muscles for emg data acquisition , as described by zipp15 . the electrodes were secured with surgical tape and a cloth wrap to minimize disruption during the movement . a ground electrode was placed on the wrist on the side opposite to the measured upper extremity . acceleration was recorded with an accelerometer attached to the measured wrist ( the dorsal midpoint between ulnar and radial styloid processes ) . the accelerometer was secured with surgical tape and a cloth wrap to minimize disruption during the movement . the x - axis was the orientation of the long axis of the forearm . verification of signal quality was completed for each muscle by having the subject perform a resisted contraction in muscle test positions specific to each muscle of interest16 . for a normalization reference , emg data were collected during maximal voluntary contraction ( mvc ) in a resisted contraction in each of the manual muscle testing positions . data were sampled in two 3-second trials during manually resisted maximal contractions of each muscle . the highest value ( averaged over 1 second ) was used as the normalization reference . for all emg raw data , m - scope ( s & me co. , japan ) after band pass - filtering between 15500 hz and sampling at 1,000 hz , root mean square ( rms ) values were calculated . the mvc values were used to normalize the emg signal amplitude of the reach - to - grasp movement . the relative value was calculated by dividing the average rms of the reach - to - grasp movement by the rms of the mvc value . the notation a ( sampled at 1,000 hz ) is used to refer to the acceleration . the x - axis acceleration was analyzed from the starting point of the reach - to - grasp movement to the second zero point . the calculated percent mvc values of the four muscles were compared during the reach - to - grasp movement . comparisons of the percent mvc values of the resting seated posture and reach - to - grasp movement phases were performed using repeated - measures analysis of variance . to determine the significance of differences , tukey s test was used as a post hoc test . the paired - samples t - test was used to test differences between the dominant and non - dominant upper extremities . in all of these tests , figure 1fig . 1.patterns of acceleration ( solid line ) , estimated velocity ( dotted line ) , and muscle activation of the upper trapezius ( ut ) , anterior deltoid ( ad ) , biceps brachii ( bb ) , and triceps brachii ( tb ) muscles during a reach - to - grasp movement for a representative object . acceleration and estimated velocity of the forward movement along the long axis of the forearm are depicted . ia : increasing acceleration phase , da : decreasing acceleration phase , i d : increasing deceleration phase , dd : decreasing deceleration phase shows a representative case with corresponding acceleration curves and emg activities . the acceleration curve was essentially a diphasic complex , as already noted9 . in the present study , the simple biphasic profile of the reaching movement starting from a value of 0 acceleration ( point 0 in fig . 1 ) and returning to that from a negative value ( point e ) was analyzed . patterns of acceleration ( solid line ) , estimated velocity ( dotted line ) , and muscle activation of the upper trapezius ( ut ) , anterior deltoid ( ad ) , biceps brachii ( bb ) , and triceps brachii ( tb ) muscles during a reach - to - grasp movement for a representative object . acceleration and estimated velocity of the forward movement along the long axis of the forearm are depicted . ia : increasing acceleration phase , da : decreasing acceleration phase , i d : increasing deceleration phase , dd : decreasing deceleration phase subjects focused on accelerating the hand in order to reach peak velocity ( point a ) at approximately one - third of the duration of the movement evaluated here . after the acceleration peak , the acceleration gradually reduced to 0 ( point b ) , where the hand reached peak velocity at half of the duration of the movement . then , the subjects rapidly decelerated the hand , and after the declaration peak ( point c ) , deceleration rapidly reduced until 0 ( point e ) , where backward movement of the hand reached peak velocity at four - fifths of the duration of the movement . during the reduction phase of the deceleration , hand movement changed smoothly from the forward direction to the backward one at point d , indicating the turning point of the hand movement to the backward direction . this finding , that acceleration was still negative when the velocity value returned to 0 , was observed in all subjects . after peak velocity of the backward direction ( point e ) , increase and decrease in acceleration occurred until the next peak velocity of the forward direction ( data not shown ) . then , the change of acceleration and deceleration , followed by the change in forward and backward directions with a small time lag , were repeated several times with reduction in the lag time ; the values of the acceleration and velocity until the point of zero acceleration corresponded to that of velocity , when transportation of the hand toward the object was completed ( data not shown ) . on the basis of the acceleration profile , this reaching movement of the reach - to - grasp task could be divided into four phases : an increasing acceleration ( ia ) phase , a decreasing acceleration ( da ) phase , an increasing deceleration ( i d ) phase , and a decreasing deceleration ( dd ) phase . emg activities showed different levels and muscle activity patterns among the muscles examined here . since the pattern of each muscle activity seemed to be associated with the acceleration profile shown in fig . interestingly , the active phases of each muscle varied among the muscles examined here ( table 1table 1.comparison of the muscle activities of the upper trapezius , anterior deltoid , biceps brachii , and triceps brachii muscles in each acceleration phaserestacceleration phasedeceleration phaseiadaidddut ( % mvc)7.5 6.137.7 22.3 * 16.8 22.220.0 9.226.5 16.3ad ( % mvc)4.3 3.230.7 10.3 33.0 13.3 * 37.5 20.8 * 42.4 29.6bb ( % mvc)8.0 11.719.5 11.9 14.3 13.420.3 16.4 * 21.6 17.9tb ( % mvc)14.3 8.215.5 7.717.2 7.619.4 11.922.6 13.6data presented as mean sd . ut : upper trapezius , ad : anterior deltoid , bb : biceps brachii , tb : triceps brachii . ia : increasing acceleration , da : decreasing acceleration , i d : increasing deceleration , dd : decreasing deceleration , sd : standard deviation . * significant ( p<0.01 ) difference between rest and movement phases , respectively ; significant ( p<0.01 ) difference between ia and da ; significant ( p<0.01 ) difference between ia and i d ; significant ( p<0.01 ) difference between ia and dd ; significant ( p<0.01 ) difference between da and i d ; significant ( p<0.01 ) difference between da and dd ; significant ( p<0.05 ) difference between i d and dd ) . significant increases in the upper trapezius activity were observed in both phase ia and phase dd compared with the resting position , by approximately 5- and 3.5-fold , respectively ( table 1 ) . furthermore , the activity was gradually augmented and reached approximately 10 times that of the resting position ( table 1 ) . significant increases in the biceps brachii activity were observed in phases ia , i d , and dd ( table 1 ) , by approximately 2.5-fold . a significant increase in the triceps brachii muscle activity there was little difference between the muscle activities of the backward and forward movements in phase dd ( data not shown ) . ut : upper trapezius , ad : anterior deltoid , bb : biceps brachii , tb : triceps brachii . ia : increasing acceleration , da : decreasing acceleration , i d : increasing deceleration , dd : decreasing deceleration , sd : standard deviation . * significant ( p<0.01 ) difference between rest and movement phases , respectively ; significant ( p<0.01 ) difference between ia and da ; significant ( p<0.01 ) difference between ia and i d ; significant ( p<0.01 ) difference between ia and dd ; significant ( p<0.01 ) difference between da and i d ; significant ( p<0.01 ) difference between da and dd ; significant ( p<0.05 ) difference between i d and dd percent mvcs of the dominant and non - dominant upper extremities were compared in the phases in which the muscles were significantly active compared to the resting position ( table 2table 2.comparison of percent mvc of the dominant and non - dominant upper extremities in the phases in which muscle were significantly active compared to the resting position(%mvc)dominantnon - dominantutphase ia37.7 22.349.7 33.1 * phase dd26.5 16.333.3 17.3 * adphase ia30.7 10.332.6 10.7phase da33.0 13.332.0 9.9phase id37.5 20.838.6 17.6phase dd42.4 29.643.0 25.8bbphase ia19.5 11.918.5 7.9phase id20.3 16.426.2 14.8 * phase dd21.6 17.935.8 23.8 * tbphase dd22.6 13.630.1 33.6data presented as mean sd . ut : upper trapezius , ad : anterior deltoid , bb : biceps brachii , tb : triceps brachii , ia : increasing acceleration , da : decreasing acceleration , i d : increasing deceleration , dd : decreasing deceleration , sd : standard deviation . * significant ( p<0.05 ) difference between percent mvc of dominant and non - dominant upper extremities ) . these comparisons found there were significant differences between the dominant and non - dominant upper extremities in phases ia and dd for the upper trapezius activity , and in phases i d and dd for the biceps brachii activity . the percent mvcs of the non - dominant upper extremity in each phase of these muscles were all larger than those of the dominant extremity . data presented as mean sd . ut : upper trapezius , ad : anterior deltoid , bb : biceps brachii , tb : triceps brachii , ia : increasing acceleration , da : decreasing acceleration , i d : increasing deceleration , dd : decreasing deceleration , sd : standard deviation . * significant ( p<0.05 ) difference between percent mvc of dominant and non - dominant upper extremities analysis showed the kinematic trajectories of acceleration during a simple biphasic profile of the reaching movement in a reach - to - grasp task could be divided into four phases : increasing acceleration ( ia ) phase , decreasing acceleration ( da ) phase , increasing deceleration ( i d ) phase , and decreasing deceleration ( dd ) phase . muscles around the shoulder showed different activity patterns , which were closely associated with these acceleration phases . interestingly , significantly smaller muscle activities for the dominant extremity , approximately two - thirds of those of the non - dominant one , were observed in phases ia and dd for the upper trapezius and phases i d and dd for the biceps brachii . there have been few studies evaluating the emg activity patterns of muscles around the shoulder in association with the kinematics of a reaching movement . sabatini showed the emg activities were characterized by phasic drives in some muscles around the shoulder during reaching movement , although what was related to the phasic pattern among the kinematic parameters was not elucidated10 . phases divided on the basis of the velocity trajectory , which is bell - shaped5 , might be related to the phasic drives of the muscles activities . the peak of the velocity profile was reported to divide the reaching movement into a hand acceleration phase ( positive values of tangential acceleration ) and a hand deceleration phase ( negative values of acceleration)9 , 17 . the present study showed that this hand acceleration phase is composed of phases ia and da . in the upper trapezius and biceps brachii muscles , the hand deceleration phase was composed of phase i d and a part of phase dd . similarly , in the upper trapezius and triceps brachii muscles , emg activation was observed only in phase dd . these findings indicate that the muscle activities examined here were related to the acceleration profile , but not to the velocity profile of the reaching movement in the reach - to - grasp task . these results suggest the importance of the four phases derived from the acceleration trajectory for the elucidation of the muscular mechanisms which regulate and coordinate the muscles around the shoulder in reaching movements . it is interesting and unexpected that hand movement changed smoothly from the forward to the backward direction during phase dd , the reduction phase of deceleration . similar findings that the acceleration value was still negative when the velocity value returned to 0 were reported by supuk et al9 . in the present study , several repeated cycles of acceleration and deceleration in a short period followed the period analyzed here . it is possible that there is a precise regulatory mechanism when the palm approaches the target . careful and precise examination will be needed to elucidate the possible factors contributing to the delicate kinematics operating in this late stage of the reach - to - grasp task , which might correspond to the phase of final closure of the fingers as reported by supuk et al9 . on the other hand , there was little difference in the muscular activity of backward and forward movements in phase dd , confirming that the muscle activities examined here were related to the acceleration profile , but not the velocity profile . muscle activity of the upper trapezius was more frequently present during the early acceleration phase . since the arm is not supported against gravity , a component of the upper trapezius activity is tonic , and results from holding the limb up against the force of gravity , particularly in the last period of the reaching movement10 . the muscle activity has also been shown to be characterized by phasic drives that occur at the movement onset and at the time when the object is grasped10 . the upper trapezius was active in elevating the arm at the start of reaching and in appropriately positioning the scapula during the movements11 . the upper trapezius acts to resist the downward rotation torques believed to occur via the reverse action of the glenohumeral muscles , including the deltoid , cuff muscles , and triceps brachii , acting on the scapula19 . together , these results suggest that the role of the upper trapezius activity may be to hold up the limb against the force of gravity during phase ia , and reverse the action of the deltoid and triceps brachii muscles for fine control of transportation of the hand toward the object by stabilizing the scapula in phase i d . the muscle may also play a reverse role to the deltoid muscle and biceps brachii in scapular stabilization in phase ia . the anterior deltoid was activated and gradually augmented over the whole duration of the movement . the anterior deltoid has been shown to be active to maintain a certain degree of shoulder abduction and to anteflex the shoulder during reaching movements11 . the deltoid muscle activity reaches an initial peak at 110 degrees when the arm is fully elevated above the head20 . it is conceivable that anterior deltoid muscle may produce shoulder flexion force with increasing shoulder moment to suspend the upper arm against the force of gravity , which may result in the continuous increase in its muscle activity seen throughout the reaching movement . the biceps brachii is active in lifting and holding the lower arm above the table and aids the anteflexion of the shoulder11 . in the elbow flexors , electrical neuromuscular stimulation of the biceps thus , the biceps brachii may lift and hold the lower arm above the table during the early acceleration phase . it is interesting that the muscle was not activated in phase da , suggesting that the moment of lifting the forearm may not be due to muscle activity but to inertia in this phase . on the other hand , the muscle was active in the deceleration phase , the phase when braking of the movement s velocity occurs . the biceps brachii muscle s activity may play a role in braking elbow extension , resulting in reduction in the speed of reaching . the muscle activity of the triceps brachii has been shown to be very low , sometimes nearly absent , in the extension of the elbow towards the target11 . it should be noted that the precise muscle regulatory mechanism , when the palm closely approaches the target with repeated forward and backward movements , may start in phase dd . therefore , it is possible that the purpose of the triceps brachii activity may be fine control of transportation of the hand , coordinated with the antagonistic biceps brachii , close to the object during phase dd . the possible roles of the muscles examined here may be summarized in each phase as follows : the upper trapezius elevates and stabilizes the shoulder girdle , the anterior deltoid produces the moment of shoulder flexion , which initiates forward movement of the upper arm , and the biceps brachii lifts the lower arm above the table during phase ia ; in phase da , the anterior deltoid keeps producing the power for shoulder flexion with increasing shoulder moment to suspend the upper arm against the force of gravity , while the other muscles are inactive , suggesting reduction of acceleration ; in phase i d , the biceps brachii acts as a braking mechanism by resisting elbow extension . finally , in phase dd , the biceps brachii acts as a brake , the anterior deltoid is still active to keep the upper arm suspended against the force of gravity , and in response to these muscles activities , the triceps brachii is activated to achieve delicate and fine control of transportation of the hand toward the object when it is very close to it , resulting in the reduction of deceleration . in response to these glenohumeral muscles activities , upper trapezius activity is induced to stabilize the scapula against downward movement . significantly smaller muscle activities of the upper trapezius and biceps examined the control of movement in the dominant and non - dominant hands , and reported that non - dominant arm motor output was subject to increased variability , which necessitated a greater number of corrective movements12 . recent research investigating the dominant arm advantage of the finger in 15-cm - long movements on a table has indicated that the dominant arm consistently uses more torque - efficient patterns for movements that were made with similar speeds and accuracy than the non - dominant arm movements , since muscle torque impulses were substantially lower at the shoulder of the dominant arm14 . the authors proposed that distinct neural control mechanisms are employed for dominant and non - dominant arm movements14 . this suggests that the dominant arm may use more torque - efficient patterns in both the shoulder and elbow joints than the non - dominant arm . on the other hand , it is unclear why muscle activities of the upper trapezius in the ia and dd phases , and the biceps brachii in the i d and dd phases , reduced in association with the putative neural control mechanism . the usual roles of these muscle activities may be the regulation of arm reaching , action to counter glenohumeral activity , and braking action against forward movement of the arm , respectively . bagesteiro and sainburg demonstrated a significant difference in direction error and deviation from linearity between dominant and non - dominant arm movements14 , implying excessive motion in non - dominant arm movement . over - activation of regulatory muscles such as the upper trapezius and biceps brachii may be elicited by responding to possibly inefficient direction error , but not a less efficient torque strategy of the non - dominant arm . to elucidate the correlation between velocity and acceleration precisely , direct measurement of velocity and acceleration will be necessary . also , the present study focused on only four muscles around the shoulder . other muscles should be examined on the basis of the acceleration phases presented here so that the role of each muscle in concert with the others can be elucidated .
backgroundthe goeckerman regimen remains one of the oldest , most reliable treatment options for patients with moderate to severe psoriasis . goeckerman therapy currently consists of exposure to ultraviolet b light and application of crude coal tar . the details of the procedure can be confusing and challenging to understand for the first - time patient or provider.objectiveto present a freely available online guide and video on goeckerman treatment that explains the regimen in a patient - oriented manner.methodsthe goeckerman protocol used at the university of california san francisco psoriasis and skin treatment center as well as available information from the literature were reviewed to design a comprehensive guide for patients receiving goeckerman treatment.resultswe created a printable guide and video resource that covers the supplies needed for goeckerman regimen , the treatment procedure , expected results , how to monitor for adverse events , and discharge planning.conclusionthis new resource is beneficial for prospective patients planning to undergo goeckerman treatment , healthcare providers , and trainees who want to learn more about this procedure . online media and video delivers material in a way that is flexible and often familiar to patients .	the goeckerman regimen is a unique combination therapy of ultraviolet b ( uvb ) light and application of crude coal tar ( cct ) for the treatment of psoriasis [ 1 , 2 ] . first introduced in 1925 , the goeckerman regimen remains one of the oldest , most reliable treatment options for patients with moderate to severe psoriasis . the advantage in using tar and phototherapy together is that tar is a photosensitizer and when combined with uvb light acts synergistically to produce better results than either treatment alone [ 35 ] . in comparison to other treatment modalities such as internal biologic agents , oral systemic agents , and topical medications , this makes goeckerman regimen an excellent alternative for patients who may have previously failed multiple therapies , the elderly , pregnant patients , children , and the immunosuppressed . goeckerman therapy was originally administered at an inpatient hospital facility for 24 h a day for multiple days until the psoriasis cleared . however , patients today are often treated in an outpatient day care setting where they return home at the end of the treatment day with similar results but significantly reduced cost [ 6 , 7 ] . currently , goeckerman therapy at the university of california san francisco ( ucsf ) psoriasis center requires a minimum time commitment of 45 h in the daycare facility for 5 days a week for 6 weeks for a total of 30 total treatment days . the major limitation of this therapy is the time commitment required as patients should avoid interruption in their therapy , which can delay complete treatment of their psoriasis lesions and shorten the remission time . nevertheless , almost all goeckerman patients have seen significant improvement in their skin condition over the duration of their therapy . in a study performed at the university of california san francisco ( ucsf ) psoriasis and day care center 100% of patients receiving goeckerman over a 12 week period achieved a 75% or greater improvement in their psoriasis lesions . another advantage of goeckerman is the long period of remission following completion of therapy , which can last between 8 months to over a year [ 6 , 9 ] . studies have also shown that goeckerman therapy can significantly increase patient satisfaction and improve overall quality of life . in addition , ucsf has developed a modified goeckerman regimen to treat other skin diseases such as eczema , prurigo nodularis , and pruritus [ 11 , 12 ] . for the first - time patient or referring provider the details of the goeckerman procedure can be confusing and challenging to understand . therefore , the following guide and online media attempt to deliver the material in a way that is easily understandable and readily accessible . below we will describe supplies for goeckerman therapy , evaluation and preparation , treatment procedure , daily assessment , discharge planning , and safety considerations . we reviewed the goeckerman therapy protocol used at the ucsf psoriasis and skin treatment center . in addition pubmed database was searched using the term psoriasis combined with the term goeckerman therapy , tar therapy , or tar and light therapy to identify relevant articles to design a comprehensive guide for patients receiving goeckerman treatment . this article does not contain any new studies with human or animal subjects performed by any of the authors . the guide below will cover the supplies needed for goeckerman regimen , the treatment procedure , how to monitor for side effects , daily assessment , and discharge planning . cct , commonly referred to as black tar , is compounded in white petrolatum ointment or cetaphil cream ( galderma laboratories , l.p . ) in concentrations of 2% , 5% , and 10% . liquor carbonis detergens ( lcd ) , commonly referred to as gold tar , is compounded in aquaphor ointment ( beiersdorf , inc . ) , cetaphil cream , or vanicream lotion ( pharmaceutical specialties , inc . ) as 20% concentration . topical steroids include clobetasol propionate 0.05% ointment , triamcinolone 0.01% ointment , and desonide 0.05% ointment . moisturizing lotion used after therapy consists of aquaphilic ointment ( medco lab inc . ) or vanicream . gown , gloves , socks , and shower cap are used for covering the skin ( table 1).table 1supplies for goeckerman regimencrude coal tar ( 2% , 5% , 10% ) gold taraquaphor ointmentcetaphil creamvanicream lotiontopical corticosteroidsclobetasol propionate 0.05% ointmenttriamcinolone 0.01% ointmentdesonide 0.05% ointmenttar removalmineral oilsoapmoisturizing lotionaquaphilic ointmentvanicreamocclusionplastic wrapmiscellaneousgownglovessocksshower cap supplies for goeckerman regimen prior to therapy a complete history and physical examination is performed for each patient to obtain important information on current and past medications , response to previous psoriasis therapies , history of adverse reactions to sunlight or phototherapy , and severity of itch . an initial assessment of the skin will help to determine the degree and severity of psoriasis involvement and whether patients display widespread or intense erythema . in cases of severe psoriasis it is recommended that patients undergo a cool down period during which topical corticosteroids will be applied to the affected areas and occluded with plastic wrap until the erythema is greatly reduced ( 314 days ) [ 1 , 11 ] . this is because uv light and tar preparations have the potential to worsen acutely inflamed psoriasis ( table 2).table 2evaluation and preparationobtain history and physicalassess for degree and severity of skin involvementperform optional cool down period evaluation and preparation after the cool down procedure ( if needed ) , the nurse will administer phototherapy in the morning each day before applying tar to affected areas of the skin . narrowband uvb is most commonly used although broadband uvb may be used in certain situations where narrowband uvb is ineffective or not tolerated by the patient [ 5 , 8 ] . a nurse will then adjust subsequent phototherapy dosing according to the patient s response and tolerance to phototherapy , noting any signs of skin burning or itching ( table 3).table 3treatment procedurepatient checks in at front desknurse administers uvb phototherapypatient asked to undress for tar applicationnurse applies cct to body and extremities and lcd to the scalptar is occluded with plastic wrappatient dressed in gown with gloves , socks , and shower captherapy continues for 45 htar is washed off with mineral oil and soapnurse applies lcd to the body before patient leaves for home cct crude coal tar , lcd liquor carbonis detergens , uvb ultraviolet b cct crude coal tar , lcd liquor carbonis detergens , uvb ultraviolet b after phototherapy , cct will be applied to affected areas of the body and lcd for scalp involvement . tar is typically started with the lowest concentration available and increased gradually as tolerated by the patient . some patients may not tolerate cct or lcd in aquaphor base due to the greasy texture or alcohol content [ 11 , 13 ] . for these patients , tar in cetaphil cream , a water based moisturizer - may be substituted . a formulation of tar compounded with salicylic acid is often used in areas with greatly thickened plaques to help reduce the scaling . however , salicylic acid should be used with caution in patients with diabetes or gastric ulcers due to the potential for adverse side effects . occlusion of topical tar is then performed with plastic wrap to the body , arms , and legs , impermeable gloves for the hands , socks for the feet , and a shower cap for the scalp ( fig . 1 ) . topical tar is typically left on the skin for a minimum of 45 h each day . during this time , patients may read , listen to music , work on a laptop computer , socialize with other goeckerman patients , or participate in group activities such as meditation , board games , or cooking ( fig . 2 ) . after the 45 h period , the tar is washed off in the shower with mineral oil and soap . after goeckerman therapy is completed at the daycare center a nurse will apply lcd in aquaphor ointment or cetaphil cream ( less greasy option ) to the body before the patient leaves for home.fig . middle row 20% liquid carbonis detergens in vanicream lotion , 20% liquid carbonis detergens in aquaphor ointment , 5% crude coal tar in white petrolatum ointment , triamcinolone 0.01% ointment . b patient spends 45 h in waiting area during tar therapy supplies for goeckerman regimen . left side gown , socks , shower cap . middle row 20% liquid carbonis detergens in vanicream lotion , 20% liquid carbonis detergens in aquaphor ointment , 5% crude coal tar in white petrolatum ointment , triamcinolone 0.01% ointment . bottom row desonide 0.05% ointment , clobetasol propionate 0.05% ointment treatment procedure . a nurse occluding tar with plastic wrap . b patient spends 45 h in waiting area during tar therapy each day the patient will be assessed by the care team consisting of a doctor , fellow , and nurse for response to goeckerman treatment . signs of skin burn such as redness of the skin , skin tightness , pain , and rarely blistering may indicate too high a dose or intolerance to light therapy . if a burning sensation develops , the phototherapy dose is either decreased or phototherapy treatment is not given at all for that day . itch is another common concern as skin irritation may be a sign of sensitivity from tar or plastic wrap , in which case the concentration of tar may be decreased or the wrap may not be applied . patients are also encouraged at this time to update their care team about any change in their medical conditions that may have occurred or outside doctor s visits they may have had ( table 4).table 4daily assessmentteam of doctor , fellows , and nurses evaluate patient response to treatmentcheck for signs of skin burn and itchadjust phototherapy dosing and tar concentration as appropriate upon completion of the goeckerman course , patients will be started on a maintenance program which may include outpatient phototherapy three times a week for the first month with gradual taper and topical medications to be applied at home . one month after completion of goeckerman the patient will be scheduled for a follow - up visit with the doctor ( table 5).table 5discharge planninghome maintenance program with outpatient phototherapy , lcd , and topical steroidsreassess patient response to therapy after 1 month lcd liquor carbonis detergens lcd liquor carbonis detergens the safety profile of goeckerman therapy is excellent with relatively few side effects . however , many studies including a review of 13,200 patients undergoing goeckerman regimen for psoriasis and eczema showed that there is no increased risk of cancer with tar therapy compared to topical corticosteroids . in addition , goeckerman therapy is entirely topical and has limited internal absorption , so it does not increase the risk for cardiovascular disease , tuberculosis , or serious infections that may be associated with some oral or injectable medications . the most commonly observed side effects include mild folliculitis , a skin condition characterized by itchy red bumps that develop around hair follicles , and mild skin burning from the uvb light . for this reason it is recommended that patients avoid extended periods of sun exposure when tar is applied at home ( table 6).table 6safety considerationsside effectsigns / symptomsskin burning redness , tenderness , pain , tightness , itching , rarely blisteringnoticeable 46 h after treatment with uvb phototherapyfolliculitisitchy red bumps that develop around hair folliclesitchingskin irritation from tar or occlusion uvb ultraviolet b patients should avoid extended periods of sun exposure when tar is applied at home safety considerations patients should avoid extended periods of sun exposure when tar is applied at home the guide below will cover the supplies needed for goeckerman regimen , the treatment procedure , how to monitor for side effects , daily assessment , and discharge planning . cct , commonly referred to as black tar , is compounded in white petrolatum ointment or cetaphil cream ( galderma laboratories , l.p . ) in concentrations of 2% , 5% , and 10% . liquor carbonis detergens ( lcd ) , commonly referred to as gold tar , is compounded in aquaphor ointment ( beiersdorf , inc . ) , cetaphil cream , or vanicream lotion ( pharmaceutical specialties , inc . ) as 20% concentration . topical steroids include clobetasol propionate 0.05% ointment , triamcinolone 0.01% ointment , and desonide 0.05% ointment . moisturizing lotion used after therapy consists of aquaphilic ointment ( medco lab inc . ) or vanicream . gown , gloves , socks , and shower cap are used for covering the skin ( table 1).table 1supplies for goeckerman regimencrude coal tar ( 2% , 5% , 10% ) black tarwhite petrolatum ointmentcetaphil creamliquor carbonis detergens ( 20% ) gold taraquaphor ointmentcetaphil creamvanicream lotiontopical corticosteroidsclobetasol propionate 0.05% ointmenttriamcinolone 0.01% ointmentdesonide 0.05% ointmenttar removalmineral oilsoapmoisturizing lotionaquaphilic ointmentvanicreamocclusionplastic wrapmiscellaneousgownglovessocksshower cap supplies for goeckerman regimen prior to therapy a complete history and physical examination is performed for each patient to obtain important information on current and past medications , response to previous psoriasis therapies , history of adverse reactions to sunlight or phototherapy , and severity of itch . an initial assessment of the skin will help to determine the degree and severity of psoriasis involvement and whether patients display widespread or intense erythema . in cases of severe psoriasis it is recommended that patients undergo a cool down period during which topical corticosteroids will be applied to the affected areas and occluded with plastic wrap until the erythema is greatly reduced ( 314 days ) [ 1 , 11 ] . this is because uv light and tar preparations have the potential to worsen acutely inflamed psoriasis ( table 2).table 2evaluation and preparationobtain history and physicalassess for degree and severity of skin involvementperform optional cool down period evaluation and preparation after the cool down procedure ( if needed ) , the nurse will administer phototherapy in the morning each day before applying tar to affected areas of the skin . narrowband uvb is most commonly used although broadband uvb may be used in certain situations where narrowband uvb is ineffective or not tolerated by the patient [ 5 , 8 ] . a nurse will then adjust subsequent phototherapy dosing according to the patient s response and tolerance to phototherapy , noting any signs of skin burning or itching ( table 3).table 3treatment procedurepatient checks in at front desknurse administers uvb phototherapypatient asked to undress for tar applicationnurse applies cct to body and extremities and lcd to the scalptar is occluded with plastic wrappatient dressed in gown with gloves , socks , and shower captherapy continues for 45 htar is washed off with mineral oil and soapnurse applies lcd to the body before patient leaves for home cct crude coal tar , lcd liquor carbonis detergens , uvb ultraviolet b cct crude coal tar , lcd liquor carbonis detergens , uvb ultraviolet b after phototherapy , cct will be applied to affected areas of the body and lcd for scalp involvement . tar is typically started with the lowest concentration available and increased gradually as tolerated by the patient . some patients may not tolerate cct or lcd in aquaphor base due to the greasy texture or alcohol content [ 11 , 13 ] . for these patients , tar in cetaphil cream , a water based moisturizer - may be substituted . a formulation of tar compounded with salicylic acid is often used in areas with greatly thickened plaques to help reduce the scaling . however , salicylic acid should be used with caution in patients with diabetes or gastric ulcers due to the potential for adverse side effects . occlusion of topical tar is then performed with plastic wrap to the body , arms , and legs , impermeable gloves for the hands , socks for the feet , and a shower cap for the scalp ( fig . 1 ) . topical tar is typically left on the skin for a minimum of 45 h each day . during this time , patients may read , listen to music , work on a laptop computer , socialize with other goeckerman patients , or participate in group activities such as meditation , board games , or cooking ( fig . 2 ) . after the 45 h period , the tar is washed off in the shower with mineral oil and soap . after goeckerman therapy is completed at the daycare center a nurse will apply lcd in aquaphor ointment or cetaphil cream ( less greasy option ) to the body before the patient leaves for home.fig . middle row 20% liquid carbonis detergens in vanicream lotion , 20% liquid carbonis detergens in aquaphor ointment , 5% crude coal tar in white petrolatum ointment , triamcinolone 0.01% ointment . b patient spends 45 h in waiting area during tar therapy supplies for goeckerman regimen . left side gown , socks , shower cap . middle row 20% liquid carbonis detergens in vanicream lotion , 20% liquid carbonis detergens in aquaphor ointment , 5% crude coal tar in white petrolatum ointment , triamcinolone 0.01% ointment . each day the patient will be assessed by the care team consisting of a doctor , fellow , and nurse for response to goeckerman treatment . signs of skin burn such as redness of the skin , skin tightness , pain , and rarely blistering may indicate too high a dose or intolerance to light therapy . if a burning sensation develops , the phototherapy dose is either decreased or phototherapy treatment is not given at all for that day . itch is another common concern as skin irritation may be a sign of sensitivity from tar or plastic wrap , in which case the concentration of tar may be decreased or the wrap may not be applied . patients are also encouraged at this time to update their care team about any change in their medical conditions that may have occurred or outside doctor s visits they may have had ( table 4).table 4daily assessmentteam of doctor , fellows , and nurses evaluate patient response to treatmentcheck for signs of skin burn and itchadjust phototherapy dosing and tar concentration as appropriate upon completion of the goeckerman course , patients will be started on a maintenance program which may include outpatient phototherapy three times a week for the first month with gradual taper and topical medications to be applied at home . one month after completion of goeckerman the patient will be scheduled for a follow - up visit with the doctor ( table 5).table 5discharge planninghome maintenance program with outpatient phototherapy , lcd , and topical steroidsreassess patient response to therapy after 1 month lcd liquor carbonis detergens lcd liquor carbonis detergens however , many studies including a review of 13,200 patients undergoing goeckerman regimen for psoriasis and eczema showed that there is no increased risk of cancer with tar therapy compared to topical corticosteroids . in addition , goeckerman therapy is entirely topical and has limited internal absorption , so it does not increase the risk for cardiovascular disease , tuberculosis , or serious infections that may be associated with some oral or injectable medications . the most commonly observed side effects include mild folliculitis , a skin condition characterized by itchy red bumps that develop around hair follicles , and mild skin burning from the uvb light . for this reason it is recommended that patients avoid extended periods of sun exposure when tar is applied at home ( table 6).table 6safety considerationsside effectsigns / symptomsskin burning redness , tenderness , pain , tightness , itching , rarely blisteringnoticeable 46 h after treatment with uvb phototherapyfolliculitisitchy red bumps that develop around hair folliclesitchingskin irritation from tar or occlusion uvb ultraviolet b patients should avoid extended periods of sun exposure when tar is applied at home safety considerations patients should avoid extended periods of sun exposure when tar is applied at home goeckerman therapy is both safe and effective for treatment of moderate to severe psoriasis and is an excellent treatment option . patients are encouraged to fully participate in this program by showing up daily , not missing therapy sessions , and communicating any symptoms or concerns to the care staff . with consistent adherence to therapy , the vast majority of patients can achieve total body clearing , even in the toughest areas such as the scalp , palms , and soles . this guide and the corresponding online video serves to orient and educate prospective patients planning to start goeckerman treatment , healthcare providers , and trainees who want to learn more about this procedure . john koo is a speaker for abbvie , leo , and celgene , and conducts research for amgen , janssen , novartis , photomedex , galderma , pfizer and merck . tina bhutani is an advisor for cutanea , and conducts research for abbvie , janssen , and merck . wilson liao conducts research for abbvie , janssen , novartis , and pfizer , and receives funding from the nih ( r01ar065174 , u01ai119125 ) . john koo , tina bhutani , and wilson liao have no stocks , employment or board memberships with any pharmaceutical company . tian hao zhu , mio nakamura , benjamin farahnik , michael abrouk , rasnik k. singh , kristina m. lee , and sarah hulse have nothing to disclose . this article does not involve any new studies of human or animal subjects performed by any of the authors . this article is distributed under the terms of the creative commons attribution - noncommercial 4.0 international license ( http://creativecommons.org/licenses/by-nc/4.0/ ) , which permits any noncommercial use , distribution , and reproduction in any medium , provided you give appropriate credit to the original author(s ) and the source , provide a link to the creative commons license , and indicate if changes were made .
quorum sensing ( qs ) is a key regulator of virulence factors and biofilm formation in gram - negative bacteria such as pseudomonas aeruginosa . microorganisms that inhabit soil are of strategic importance in the discovery of compounds with anti - qs properties . the objective of the study was to test the culture extract of a taxonomically novel species of paenibacillus strain 139si for its inhibitory effects on the qs - controlled virulence factors and biofilm formation of pseudomonas aeruginosa both in vitro and in vivo . the paenibacillus sp . culture extract was used to test its anti - qs effects on the lasa protease , lasb elastase , pyoverdin production , and biofilm formation of p. aeruginosa as well as evaluate its therapeutic effects on lung bacteriology , pathology , hematological profile , and serum antibody responses of experimental animals in a rat model of chronic lung infection . results showed significant decrease in the activities of qs - controlled lasa protease , lasb elastase pyoverdin , and biofilm formation of p. aeruginosa caused by the culture extract . moreover , the extract significantly prolonged the survival times of rats and facilitated the clearance of biofilm infections from infected lungs . in conclusion , the antiquorum sensing effects of culture extract from a novel species of paenibacillus provide new insights to combat biofilm - associated infections .	quorum sensing ( qs ) is a key regulator of virulence factors and biofilm formation in gram - negative bacteria such as pseudomonas aeruginosa . this qs system comprises a signal molecule , a synthetase to make this signal , and a regulator to regulate gene expression . several signaling molecules have been identified ; however , the main molecules produced by gram - negative bacteria are acylhomoserine lactones ( ahls ) . it has been reported that bacterial biofilms are associated with chronic infections such as cystic fibrosis ( cf ) and tonsillitis . the discovery of qs system and its critical role in bacterial virulence has revealed new targets to attenuate their pathogenicity . there are a number of ways to interrupt the qs system , one of which is the use of microbial natural products which represent an important step towards the discovery of novel therapeutic chemicals [ 5 , 6 ] . despite the fact that soil is arguably the most useful and valuable habitat on earth , it is still considered one of the least understood ecosystems that needs to be further explored . soil is a major source of bacteria that synthesize a wide range of compounds with versatile biological effects [ 8 , 9 ] . , there are 160 species of paenibacillus approved and validated according to the bacterial nomenclature list by dsmz . therefore , interest in paenibacillus spp . as a source of new antimicrobial agents is increasing . advances in medical practice have led to the proper management of acute bacterial infections . however , the efficiency of many antibiotics is currently decreasing due to the occurrence of multidrug resistant bacteria . pathogenic strains of p. aeruginosa possess the ability to form biofilms which contribute to its reduced susceptibility towards antibiotics and ability to cause chronic infections . since virulence factors and biofilm formation in gram - negative bacteria are under the control of quorum sensing system , thus discovery of anti - qs compounds can be of great interest in the treatment of biofilm - associated chronic infections . moreover , the use of animal models is essential to gain a better understanding of the mechanisms involved in biofilm formation . this approach is usually accomplished by infecting a vertebrate animal with the organism of choice followed by evaluation of the animal 's immune responses . in this study , culture extract from a taxonomically novel species of paenibacillus isolated from an agricultural soil in malaysia was tested for its qs inhibitory effects in vitro on lasa protease , lasb elastase , pyoverdin production , and biofilm formation of p. aeruginosa and evaluated for its antibiofilm therapeutic effects in vivo on lung bacteriology , lung pathology , hematological profile , and serum antibody responses in vivo using a rat model of chronic biofilm - associated lung infection . the strain 139si ( genbank accession number : jf825470.1 ) from three strains of paenibacillus isolates previously isolated from an agricultural soil in malaysia was chosen as the type strain of the selected novel species . these strains were identified as members of the genus paenibacillus on the basis of phenotypic characteristics , phylogenetic analysis , and 16s rrna g+c content . the taxonomically novel species of paenibacillus strain 139si was deposited at the american type culture collection ( atcc ) with a cataloguing number ( atcc - baa-2268 ) . the strain was used to prepare the culture extract to examine its anti - qs inhibitory effects in vitro and in vivo . upon approval from the medical ethics committee at university malaya medical centre ( ummc ) ( ppum / upp/300/02/02 ref . number 744.11 ) , a clinical isolate of pseudomonas aeruginosa was collected from the palatine tonsils of a patient undergoing elective tonsillectomy at ummc . the isolate was identified via colony morphology , culturing on selective media and biochemical tests followed by assessment of its antibiotic susceptibility via disk diffusion where the isolate was shown to be multidrug resistant . the isolate was then used as the test strain in the preparation of test supernatant for lasa protease , lasb elastolytic , pyoverdin , and biofilm formation assays in vitro as well as the challenge strain in the rat model of chronic lung infection in vivo . reference strain for the lasa assay included staphylococcus aureus ( atcc 25923 ) whereas reference strains for the biofilm formation assay included pseudomonas aeruginosa ( atcc 27853 ) and escherichia coli ( atcc 25922 ) . for the lasa protease , lasb elastolytic , pyoverdin , and biofilm formation assays as well as the rat model of chronic lung infection , commercially available anti - qs compound 2(5h)-furanone 98% ( sigma - aldrich ) was used as the positive control . furanones act by mimicking the ahl signal produced by gram - negative bacteria , presumably by occupying the binding site on the putative regulatory protein , rendering it highly unstable and accelerating its turnover rate and thus resulting in the rapid disruption of the quorum sensing - mediated gene regulation . for the rat model of chronic lung infection , commercially available sodium alginate powder ( sigma - aldrich ) , that is , alginic acid sodium salt derived from brown algae , was used as the main component of the biofilm - alginate produced similarly by the live mucoid strains of p. aeruginosa to embed the challenge bacterial strain to closely resemble a lung infection caused by biofilms . in a previous study , the culture extract of paenibacillus sp . strain 139si was tested for its oral acute toxicity and was reported as nontoxic . in our study , the extract will be tested for its qs inhibitory effect in vitro and therapeutic effects in vivo . a single colony from the novel paenibacillus sp . strain 139si was transferred into sterile brain heart infusion ( bhi ) broth ( bd difco ) and incubated for 72 hours at 37c to allow maximum secretion of bioactive secondary metabolites into the culture media . the culture was then transferred aseptically into 50 ml conical bottom centrifuge tube ( jet bio fil ) and centrifuged at 8000 rpm for 20 min at 4c to separate cell from supernatant . the obtained cell - free supernatant was then subjected to sterile filtration to remove unwanted particles using syringe filter with a pore size of 0.22 m ( minisart sartorius ) . the sterile supernatant underwent freeze - drying ( lyophilisation ) and was then dissolved in ultra - pure water . the prepared stock was stored at 80c to be used for in vitro and in vivo experiments . for lasa protease , lasb elastolytic , and pyoverdin assays , a stationary - phase overnight culture of pseudomonas aeruginosa clinical isolate was grown in lb medium at 37c with shaking . the culture was then diluted 100-fold in lb medium and allowed to grow to an optical density at 600 nm ( od600 ) . at this point , culture was divided into 10 ml aliquots and an additional volumes of 1 ml and 4.5 ml of paenibacillus sp . culture extract was added to final concentrations of 1 mg / ml and 4.5 mg / ml . the lasa staphylolyticprotease activity was measured by determining the ability of p. aeruginosa test supernatant to lyse boiled cells of staphylococcus aureus . a 30 ml volume of an overnight s. aureus culture was boiled for 10 min and then centrifuged for 10 min at 10,000 g ( 13,000 rpm ) . the resulting pellet was resuspended in 10 mm na2po4 ( ph 4.5 ) to an od600 of 0.5 mcfarland standards . optical density of the mixture ( od600 ) was determined after 5 , 10 , 20 , 30 , 45 , and 60 min . activity was expressed as the change in od600 per hour per g protein [ 1 , 2 ] . the elastolytic activity of the p. aeruginosa test supernatant was determined by using elastin congo red ( ecr ) ( sigma - aldrich ) as described previously . briefly , a 100 l aliquot of test culture was added to 900 l of ecr buffer ( 100 mm tris , 1 mm cacl2 , ph 7.5 ) . insoluble ecr was removed by centrifugation , and absorption of the p. aeruginosa test supernatant was measured at a wavelength of 495 nm . activity was expressed as the change in the od495 nm per g protein [ 1 , 2 ] . the pyoverdin assay was adapted from the methods of cox and adams . briefly , the prepared p. aeruginosa test supernatant was diluted 10-fold in tris - hcl buffer ( ph 7.4 ) , and 100 l aliquots were added to 96-well microtiter plates on ice . pyoverdin concentration was based on the fluorescence of the test supernatant at an excitation wavelength of 405 nm and an emission wavelength of 465 nm using a microtiter absorbance reader ( imark , bio - rad ) . although pyoverdin is considered a marker of quorum sensing , a drop in its production may be due to an indirect effect via ph or iron concentration changes . to eliminate the chance of false - positive results strain 139si culture extract on the attachment phase of biofilm formation was measured using microtiter plate ( mtp ) assay as described previously [ 27 , 28 ] . briefly , a 180 l aliquot of sterile lb broth and a 150 l aliquot of overnight culture of p. aeruginosa isolate were transferred into the 96-well microtiter plates . culture extract with final concentrations of 1 mg / ml and 4.5 mg / ml and then incubated for 24 hours at 37c . wells were washed three times with phosphate - buffered saline to remove the weakly adherent cells and were allowed to air - dry prior to staining . the adherent biofilms were stained with a 200 l aliquot of 0.4% crystal violet solution ( w / v ) for 10 min . this was followed by adding a standard solution of 95% ethanol to extract ( solubilize ) the crystal violet from the stained biofilms . optical density of biofilms was measured at a wavelength of 570 nm ( od570 ) using a microtiter absorbance reader ( imark , bio - rad ) . to compensate for possible differences in growth rates under different incubation conditions , the adherence index was adjusted as an estimate of the density of biofilm which would be generated by a culture with an od600 of 0.5 mcfarland standards . experiment was performed in triplicate and the data was then averaged and standard deviation was calculated . calculation of the adherence index was done according to the following formula : adherence index = mean density of biofilm ( od570 ) 0.5/mean growth ( od600 ) . the guidelines for care and use of laboratory animals were followed according to the national academy of sciences . experimental protocol was approved by the animal ethics committee at the faculty of medicine , university of malaya ( pm/27/07/2010/maa ( r ) ) . a total of 48 adult sprague dawley ( sd ) rats including 24 males and 24 females were obtained from the animal care unit center ( acuc ) . rats weighing 150200 gm were kept in wire - bottomed cages at a 25c temperature in a 12-hour light - dark cycle . furthermore , general observations were recorded on the basis of behavioral changes such as food intake , salivation , muscular weakness , reflexes , and locomotion . rats were sacrificed under anesthesia with an intramuscular combination of ketamine and xylazine ( 1 ml of 100 mg / ml xylazine + 9 ml of 100 mg / ml ketamine ) given at a dose of 0.1 ml/100 gm of body weight . on day 7 after infection , randomly selected rats from each group were sacrificed and their initial macroscopic and microscopic evaluation of acute inflammation was carried out . a longer observation period was chosen to allow lung abscess formation and a second examination was carried out on day 15 for all the surviving rats to evaluate their general observations and macroscopic and microscopic lung pathology . briefly , p. aeruginosa strain was cultured in 80 ml of bhi broth for 24 hours at 37c with shaking ( 170 rpm ) . cells were centrifuged for 30 min at 10,000 g ( 20,000 rpm ) at 4c and resuspended in 2 ml of fresh bhi broth and colony forming unit ( cfu ) was counted . to mimic the biofilm 's environment , p. aeruginosa strain was immobilized in a solution of sodium alginate as described previously [ 34 , 35 ] . alginate powder was dissolved in normal saline to a concentration of 10 mg / ml and then autoclaved . a volume of 1 ml inoculation was mixed with 9 ml of sterile alginate . rats were randomly divided into eight groups , with males in groups 14 and females in groups 58 , as the following : negative control ( groups 1 and 5 ) , normal saline ( 5 ml / kg , orally ) daily + sterile distilled water ( 1 ml / kg , orally ) ( daily for 14 days ) ; positive control ( groups 2 and 6 ) , challenge bacterial inoculation ( 1 10 cfu / rat ) ( 5 ml / kg , intratracheally ) ( twice at day 1 and 2 + sterile distilled water ( 1 ml / kg , orally ) daily for 14 days ) ; comparative control ( groups 3 and 7 ) , challenge bacterial inoculation ( 1 10 cfu / rat ) ( 5 ml / kg , intratracheally ) ( twice at day 1 and 2 + 2(5h)-furanone ( 25 gm / kg , orally ) daily for 14 days ) ; extract treatment ( groups 4 and 8) , challenge bacterial inoculation ( 1 10 cfu / rat ) ( 5 ml / kg , intratracheally ) ( twice at day 1 and 2 + paenibacillus sp . extract at concentration 4.5 mg / ml ( 25 gm / kg , orally ) daily for 14 days ) . negative control ( groups 1 and 5 ) , normal saline ( 5 ml / kg , orally ) daily + sterile distilled water ( 1 ml / kg , orally ) ( daily for 14 days ) ; positive control ( groups 2 and 6 ) , challenge bacterial inoculation ( 1 10 cfu / rat ) ( 5 ml / kg , intratracheally ) ( twice at day 1 and 2 + sterile distilled water ( 1 ml / kg , orally ) daily for 14 days ) ; comparative control ( groups 3 and 7 ) , challenge bacterial inoculation ( 1 10 cfu / rat ) ( 5 ml / kg , intratracheally ) ( twice at day 1 and 2 + 2(5h)-furanone ( 25 gm / kg , orally ) daily for 14 days ) ; extract treatment ( groups 4 and 8) , challenge bacterial inoculation ( 1 10 cfu / rat ) ( 5 ml / kg , intratracheally ) ( twice at day 1 and 2 + paenibacillus sp . extract at concentration 4.5 mg / ml ( 25 gm / kg , orally ) daily for 14 days ) . upon sacrifice , the thoracic cavity of rats was opened by an excision through the peritoneum and lung specimens were harvested and cut into two parts . unfixed lungs were prepared for quantitative bacteriological evaluation as described previously [ 14 , 37 ] . briefly , lungs were stored for 2 hours at 4c and were mixed with 5 ml of cold sterile phosphate - buffered saline at 4c . diluted homogenized samples were plated on nutrient agar plates to determine the bacterial colony forming unit ( cfu ) per lung after incubation for 24 hours at 37c . the qualitative analysis of macroscopic lung pathology including abscess , consolidation , atelectasis , and hemorrhage was expressed as the lung index of macroscopic pathology ( limp ) which was calculated by dividing the area of left lung showing pathologic changes by the total area of the same lung . moreover , gross pathological changes in the lungs were assigned four different scores according to the severity of the inflammation as described previously [ 34 , 39 ] : ( i ) normal lungs ; ( ii ) swollen lungs , hyperemia , and small atelectasis ( 10 mm ) ; ( iii ) pleural adhesions and atelectasis ( 40 mm ) ; and ( iv ) abscesses , large atelectasis , and hemorrhages . lung specimens were fixed with 10% neutral buffered formalin ( nbf ) for 24 hours and processed as described previously . lungs were embedded in paraffin wax using an embedding center ( leica eg1160 , leica biosystems . germany ) and fixed onto glass slides using a water bath ( leica hi1210 , leica biosystems . tissue sections were stained with hematoxylin and eosin ( h&e ) stain and mounted with diphenyl xylene ( dpx ) to be visualized using an upright light microscope ( eclipse lv150l , nikon instruments inc . , lung pathology was assigned microscopically one of four scores according to the severity of inflammation as follows : ( 1 ) normal histology ; ( 2 ) mild focal inflammation ; ( 3 ) moderate to severe focal inflammation with areas of normal tissue ; and ( 4 ) severe inflammation to necrosis . lung cellular alterations were classified as acute or chronic inflammation [ 38 , 39 ] . slides were stained for standard light microscopy using h&e and periodic acid schiff ( pas ) stains according to the manufacturers ' instructions . to preserve the biofilm 's architecture , specimens were examined using confocal laser scanning microscope ( clsm ) coupled with double fluorescent staining as described previously . tissue sections were embedded in optimal cutting temperature ( oct ) media followed by snap freezing with a mixture of cold isopentane - liquid nitrogen to form solid blocks that were cut into a thickness of 5 m using a cryostat ( leica cm1850 , leica microsystems . each section was then fixed with 70% cold acetone for 10 min followed by double staining with 500 l propidium iodide ( pi ) for 5 min to detect bacterial cells in red followed by staining with 500 l concanavalin a ( con a ) fluorescent isothiocyanate ( fitc ) for 5 min to detect glycocalyx matrix in green . sections were then washed in a solution of phosphate - buffered saline ( pbs ) and demineralized water and were embedded in a mounting medium of pbs / glycerol containing an antiquenching agent ( p - phenylenediamine ) . carl zeiss , germany ) available at universiti putra malaysia ( upm ) , malaysia . the microscope was equipped with a krypton - argon laser for visualization of con a fitc ( number of signals acquired , 488 nm ; emission 552 df , 32 nm ) and propidium iodide ( number of signals acquired , 568 nm ; emission 605 df , 32 nm ) . digital images of the optical sections were collected using zen 2010 software and converted to high - quality jpeg files using available software . upon sacrifice , blood was drawn from the jugular vein under anesthesia . collected blood specimens were immediately transported to the clinical diagnostic laboratories at university malaya medical centre ( ummc ) . whole blood was collected using the violet caped vacuette edta tubes for differential blood count test including neutrophils , lymphocytes , monocytes , eosinophils , and basophils . serum was collected using the red caped vacuette serum tubes for immunoglobulins level in which their concentrations against p. aeruginosa standard antigen ( st - ag ) were assessed by elisa including serum antibody levels of igg , iga , igm , and ige . elisa units were obtained by dividing the mean absorbance of samples by the mean absorbance of internal standard expressing between 0 30 and 0 40 absorbance units . statistical analysis was carried out using the statistical product and service solutions software ( ibm spss statistics 21 ) . categorical data were compared by the test , while unpaired differences in continuous data were compared by both the mann - whitney u test and the analysis of variance ( anova ) test . all values were reported as standard error mean ( s.e.m ) and a probability value of ( p < 0.05 ) was considered to be statistically significant . the lasa staphylolytic protease is a zinc metalloendopeptidase belonging to the -lytic endopeptidase family of proteases . there was a significant decrease in lasa activity compared to that of the control when pseudomonas aeruginosa test supernatant was grown in the presence of paenibacillus sp . culture extract at concentrations 1 mg / ml and 4.5 mg / ml . the lb medium negative control showed no significant change in lasa activity . the lasb elastase is a zinc metalloprotease capable of destroying or inactivating a wide range of biological tissues and immunological agents . there was a significant decrease in the od readings due to the inhibitory effect by both paenibacillus sp . culture extract at a concentration of 4.5 mg / ml caused more decrease in the activities of both lasa protease and lasb elastase compared to concentration 1 mg / ml . pyoverdins are virulence factors that compete with mammalian transferrin for iron and promote pathogenicity by stimulating bacterial growth . one of the pyoverdins is suggested to be a qs - like molecule , regulating both itself and the production of other toxins . there was a significant decrease in the od readings due to the inhibitory effect by both paenibacillus sp . culture extract at a concentration of 4.5 mg / ml exhibited decreased production of the qs - controlled pyoverdin compared to concentration 1 mg / ml . all of the tested cultures retained a ph of 7.0 , regardless of the amount or the type of extract added . biofilm formation by p. aeruginosa leads to increased resistance and creates a severe infection in the lungs of patients with cystic fibrosis . there was a significant decrease in the od readings due to the inhibitory effect by both paenibacillus sp . culture extract at a concentration of 4.5 mg / ml caused decrease in the qs - controlled biofilm formation compared to concentration 1 mg / ml . culture extract significantly prolonged the survival times of experimental rats in both treatment and comparative control groups . comparative control group had its share of mortalities with 3 ( 25% ) rats on days 6 and 7 . all rats in positive control group died before completing the course of experiment ; some died from a severe infection on day 3 after inoculation whereas others died on days 5 and 10 . the greatest decrease of weight following administration of challenge p. aeruginosa strain was seen among positive control group followed by comparative then extract treatment groups . all rats , except for negative control group , were infected with a sublethal dose of challenge p. aeruginosa inoculation of 1 10 cfu / rat ( 5 ml / kg , intratracheally ) thrice at days 1 , 2 , and 3 . immediately after inoculation gm / kg , orally ) daily for 14 days whereas rats in the treatment group received paenibacillus sp . extract ( 25 gm / kg , orally ) daily for 14 days . on day 7 after infection , randomly selected rats from each group were sacrificed and their lung bacterial count was evaluated . the value of lung bacterial cfu in both comparative control and extract treatment groups dropped significantly on day 7 compared to those in positive control group . extract as well as the control compound 2(5h)-furanone started to have its inhibitory effect within 7 days of their administration . results suggested that infecting p. aeruginosa and their biofilms were cleared away more quickly in the groups receiving treatments with anti - qs compounds . on day 15 after infection , all rats were sacrificed and the median of lung bacterial cfu in extract treatment group was found to be only one - fifth of that in negative control group and the difference was significant ( p < 0.05 ) , indicating that paenibacillus sp . extract could influence the colonization and persistence of p. aeruginosa within the infected lungs . the value of lung bacterial cfu in positive control group is significantly higher than the values of the lung bacterial cfu in the rest of the groups ( p < 0.05 ) ; this was due to colonization of p. aeruginosa in the lung without their qs system being interrupted . however , the values of lung bacterial cfu among comparative control group and extract treatment group did not differ and were statistically insignificant . in agreement with the results of lung bacteriology , milder macroscopic ( gross ) lung pathology was observed in both comparative control group and extract treatment group . on day 7 after infection , the macroscopic lung pathology of positive control group showed large haemorrhage and abscess ( > 40 mm ) . a total of 2 rats died on days 3 and 5 after infection due to the severity of infection . however , the macroscopic lung pathology of comparative control group showed swollen lungs with hyperemia , small atelectasis , and moderate haemorrhage ( 10 mm40 mm ) whereas in extract treatment group the lungs were also swollen with hyperemia , atelectasis , and small haemorrhage ( < 10 mm ) . results indicated that paenibacillus sp . culture extract significantly restricted the gross pathologic changes of lung to a smaller area during the acute phase of infection . on day 15 after infection , lung abscesses and haemorrhage became predominant among positive control group . a significantly higher frequency of lung abscesses was noted in positive control group ( p < 0.05 ) than in the comparative and extract treatment groups , in which 80% of rats showed lung atelectasis and only 20% of rats showed lung abscesses . the number of rats with chronic inflammation in both comparative control group 6 ( 12.5% ) and extract treatment group 6 ( 12.5% ) was significantly lower than that in positive control group 12 ( 25% ) . intensity of lung infections on the gross pathology was more on day 15 after infection than day 7 in all groups except for negative control as there was no lung infection . results of lung index of macroscopic pathology are shown in ( figure 1 ) . on day 7 after infection , positive control group showed acute lung inflammation , multiple lung abscesses , haemorrhage , and consolidation whereas some rats died on days 3 and 5 . chronic inflammation was seen in all the 12 rats from positive control group . however , both comparative control and extract treatment groups showed chronic inflammation in 6 out of 12 rats whereas no inflammation was seen in negative control group . there were no differences in the inflammatory classification between comparative control and treatment groups . on day 15 after infection , the size of lung abscesses in comparative group was reduced compared to extract treatment group and the severity of chronic inflammation in general was decreased to half in these two groups . pathology scoring showed normal lung tissue in all the rats , 12 ( 25% ) of negative control group with score i ( figure 2(a ) ) , whereas 10 ( 20.83% ) rats in positive control group showed severe inflammation to necrosis with score iv ( figure 2(b ) ) . both comparative group 5 ( 10.4% ) and extract treatment group 6 ( 12.5% ) showed signs of recovery with score ii ( figure 2(c ) ) whereas 4 ( 8.33% ) in comparative group and 5 ( 10.4% ) in extract treatment group showed moderate to severe focal inflammation with score iii ( figure 2(d ) ) . differences in the inflammatory classification were insignificant between comparative control group 2 ( 4.16% ) and extract treatment group 1 ( 2.08% ) with score iv ( figure 2(b ) ) . moreover , extract treatment group showed much milder chronic inflammation compared to comparative control where the areas with pathologic changes were smaller . the incidence of acute inflammation was lower than that in extract treatment group ; however the differences were statistically insignificant . results of microscopic lung pathology among experimental rats are shown in table 2 and figure 2 . on day 7 after infection , sacrificed rats in positive control group showed dense biofilm layers occupying the lung alveolar spaces along with severe haemorrhage whereas some rats died on days 3 and 5 after inoculation with p. aeruginosa . moreover on day 15 after infection , surviving rats among positive group were sacrificed and their lungs showed chronic inflammation with heavily dense biofilm layers blocking and occupying ( 80% ) lung alveoli . in contrast , rats in both comparative group administered with the commercial anti - qs compound 2(5h)-furanone and extract treatment groups treated with the paenibacillus sp . culture extract showed milder inflammation with disrupted biofilm layers occupying ( 30% ) lung alveoli . there was no significant difference in biofilm density within lung tissue between rats treated with 2(5h)-furanone and those treated with the culture extract . the challenge p. aeruginosa immobilized in seaweed alginate caused mechanical blocking and damage to the alveoli and was visualized under light microscope as microcolonies embedded in alginate by using h&e stain ( figures 3(a ) and 3(b ) ) and pas stain ( figures 3(c ) and 3(d ) ) . moreover , p. aeruginosa was visualized under clsm as interconnected bacteria ( red ) encased in a scaffolding network composed of extracellular matrix ( green ) by using double florescent staining ( figure 4 ) . under clsm , microscopic examination revealed no biofilms with normal histology among rats of negative control group ( figure 4(a ) ) whereas positive control group showed intense biofilms filling the alveolar spaces with severe inflammation ( figure 4(b ) ) . both comparative control and extract treatment groups showed disrupted biofilms with mild inflammation ( figures 4(c ) and 4(d ) ) indicating the same inhibitory effect of paenibacillus sp . the intensity of biofilms occupying lung tissue was more on day 15 after infection than day 7 in both comparative control and extract treatment groups among males and females ; however differences were statistically insignificant . in agreement with the macroscopic and microscopic lung pathology , results of differential blood count ( table 3 ) and serum antibodies ( table 4 ) among negative control group showed normal levels and no significant differences ( p > 0.05 ) due to an uncompromised immune status . in contrast , positive control group showed a significant increase in neutrophils , lymphocytes , and monocytes counts as well as an increase in igm , igg , and iga levels indicating an immune response towards an infection . on day 7 after infection , both comparative control and extract treatment groups showed a marked decrease in neutrophils , lymphocytes , and monocytes as well as igm , igg , and iga levels compared to positive control group . culture extract on the infecting p. aeruginosa biofilms within lung alveoli . on day 15 after infection , positive control group showed a marked increase in differential blood count and immunoglobulins levels particularly igm and igg compared to comparative control and extract treatment group in which their igm , igg , and iga levels were lower ( p < 0.05 ) . eosinophils and basophils count ige levels in all groups were within the reference normal range . differences in differential blood account and serum antibody responses between male and female rats were statistically insignificant ( p > 0.05 ) . among the promising approaches to combat biofilm infections is the use of metabolites synthesized from different bacterial species . a large number of natural and synthetic compounds have been described exhibiting quorum sensing ( qs ) inhibitory effects against gram - negative pathogens such as pseudomonas aeruginosa . lactonase and acylase from actinobacteria and proteobacteria , coral associated bacterial extracts , and bacillus sp . these compounds function by either competing with the activity of ahl molecules due to their structural similarity or accelerating the degradation of the luxr / lasr receptors of ahl . the virulence of p. aeruginosa is mainly due to its capacity to degrade host tissue with proteases and to form biofilms . culture extract derived from a taxonomically novel species of paenibacillus strain 139si was tested for its ability to inhibit the qs - controlled virulence factors and biofilm formation of a multidrug resistant isolate of p. aeruginosa in vitro as well as its therapeutic effects in vivo using a rat model of chronic biofilm - associated lung infection . since qs is involved in virulence factor production and biofilm formation of p. aeruginosa , we expected paenibacillus sp . culture extract will have significant effects on the qs - controlled virulence factors . indeed , the extract exhibited inhibitory effect on the ability of p. aeruginosa to produce virulence factors and biofilm formation . this was similar to the findings made by adonizio et al . where they have detected anti - qs activities of six south florida medicinal plants examined against p. aeruginosa pao1 . disruption of the qs system with compounds such as halogenated furanones from the australian macroalgae delisea pulchra has also been shown to inhibit biofilm growth . therefore , in our study the compound 2(5h)-furanone 98% was used as an anti - qs control for both in vitro and in vivo experiments showing a qualitative change in biofilm morphology and a reduction in its thickness . this was similar to the findings made by hentzer et al . where they used furanones from d. pulchra showing significant inhibition in biofilm formation . there was a significant decrease in lasa activity compared to that of control when p. aeruginosa was grown in the presence of paenibacillus sp . extract at a concentration of 4.5 mg / ml ( 75% decrease ) compared to a concentration of 1 mg / ml ( 45% decrease ) ( table 1 ) . where they suggested that the compounds vescalagin and castalagin are responsible for the reduction in lasa activity . there was a significant reduction in lasb elastase activity when p. aeruginosa was grown in the presence of culture extract at a concentration of 4.5 mg / ml ( 70% decrease ) indicating that compounds in the tested extract may have downregulated the production of lasb and/or inhibited its activity . this was similar to the findings made by rasmussen et al . where they reported a 50% decrease in lasb activity using 2% garlic moreover , the control anti - qs compound , that is , 2(5h)-furanone , showed a significant decrease ( 80% ) in lasb activity similar to the findings made by hentzer et al . where they reported a 90% decrease using purified halogenated furanone from the red alga delisea pulchra . there was a significant reduction in pyoverdin production of p. aeruginosa with 65% reduction in pyoverdin levels by paenibacillus sp . this was similar to the findings made by hentzer et al . where they used synthetic derivate of natural furanones with a 90% reduction in pyoverdin levels there were no significant changes in cell growth corresponding to pyoverdin production , leaving an anti - qs effect as the most likely hypothesis . however , further studies are needed to assess the effect of paenibacillus sp . culture extract on direct enzyme inhibition of p. aeruginosa in comparison to quorum sensing signaling . inhibition of biofilm formation without influencing bacterial growth is a characteristic of antivirulence therapies which are the promising alternatives to combat bacterial infections . in our study , the use of paenibacillus sp . culture extract at a concentration of 4.5 mg / ml resulted in a significant inhibition of biofilm formationin p. aeruginosa ; however the influence of this extract on bacterial growth was insignificant . this study demonstrates that paenibacillus sp . culture extract inhibits the qs - controlled biofilm formation of p. aeruginosa on hydrophobic surfaces ( polystyrene ) as indicated by the adherence index . this was similar to the previous findings suggesting that coated clinical materials with antimicrobial and anti - qs substances can lead to a successful prevention of microbial colonization [ 61 , 62 ] . culture extract may be used as a tool to prevent microbial biofilm formation on hydrophobic and hydrophilic medicinal devices . it has been reported that the qs gene expression in p. aeruginosa is interconnected with other regulatory systems that responds to various environmental signals . the virulence factors lasa ( protease ) and lasb ( elastase ) are under the control of the lasi - lasr system ; however , the rhli - rhlr system also controls activity to a lesser extent . pyoverdin is believed to be under the control of rhli - rhlr , whereas biofilm formation is partially under the control of qs system . due to the redundancy of qs system in p. aeruginosa and the complex chemistry of metabolites produced by paenibacillus sp . culture extract , it was difficult to link the ahl level , qs gene expression , and virulence factor production . however , there was an overall inhibition of qs system in p. aeruginosa suggesting that multiple chemicals in the culture extract may have distinct qs inhibitory effects and that this effect is not directly on the las - rhl system but , rather , on a more universal qs regulator . where they have reported that the inhibitory effects of their compound maybe be due to a global qs regulator , such as vfr or gaca . more studies are needed to identify the regulated qs genes , specificity of n - acyl - homoserine lactone signals , and environmental effects on gene expression . culture extract and control 2(5h)-furanone exhibited lower bacterial numbers in lungs , indicating that the ability of challenge p. aeruginosa to colonize the lung may be reduced due to the inhibition of qs signals or because the bacterial clearance in the hosts is improved by the anti - qs effects of paenibacillus sp . consequently , the inhibition of qs attenuates the virulence of p. aeruginosa and impairs its colonization ability . these results indicates that paenibacillus sp . culture extract appears to be a promising novel antivirulent agent that possess quorum - sensing inhibition leading to increased clearance of bacteria from infected lungs and decreased lung pathology . this was similar to a recent proposal reporting that qs is a target for treatment of gram - negative bacterial infections . culture extract on bacterial clearance correlated negatively with their concentrations , which further confirmed that the action of furanones was dosage - dependent . this was similar to the findings made by wu et al . where they have reported synthetic furanones to inhibit , in a dosage - dependent manner , the qs of pseudomonas aeruginosa lung infection in mice . results showed that macroscopic lung pathology among extract treatment group was milder in comparison to positive control group . culture extract to p. aeruginosa - infected rats might induce an enhanced oxidative burst response that can aid in clearing the bacterial infection effectively and thus prolong survival time of rats with milder macroscopic lung pathology . where they reported that the administration of ginseng extract leads to the activation of polymorphonuclear leukocyte ( pmn ) which reduced bacterial load in a rat model of chronic p. aeruginosa pneumonia . may have activated the endotoxin - primed neutrophils which resulted in reduced macroscopic and microscopic lung pathology . this was similar to a previous study where they have reported that furanones successfully interfered with n - acyl homoserine lactone and suppressed bacterial qs which resulted in an accelerated clearance of p. aeruginosa in the lungs . it has been reported that inhibition of the biofilm 's alginate barrier using metabolites derived from culture extract of paenibacillus sp . may be useful in the disruption of p. aeruginosa biofilms in infected lungs of cystic fibrosis patients . in our study , it was noticed that the areas with chronic inflammatory changes in paenibacillus sp . where they have evaluated the effect of ginseng treatment on the oxidative burst response of peripheral blood neutrophils and alveolar macrophages in a rat model of chronic mucoid p. aeruginosa lung infection . the fact that majority of rats classified under score ii belong to both comparative control group 5 ( 10.4% ) and the treatment group 6 ( 12.5% ) whereas majority under score iv belong to the positive control group 10 ( 20.83% ) indicates the anti - qs effects of paenibacillus sp . we have established a rat model of chronic lung infection based on p. aeruginosa isolate with the use of artificial embedding agents . our animal experiment was accurate in developing the pulmonary infection since the number of bacteria cultured from lungs remained high throughout the infection period . furthermore , the histopathological features in rats were similar to those of chronic p. aeruginosa lung infections in humans . where they used a mucoid clinical isolate of p. aeruginosa nh57388a with hyperproduction of alginate in a mouse model of chronic lung infection . the use of alginate to embed p. aeruginosa adds a selective survival advantage for the challenge bacterial strain this was similar with a previous study where they reported improved persistence in mice lungs of mucoid p. aeruginosa strains relative to nonmucoid strains . this discrepancy may reflect differences in the virulence of p. aeruginosa strains and/or the genetic background of the animals . p. aeruginosa isolate embedded in alginate was able to establish a persistent lung infection in rats of all groups ; however the severity of infection was lower in rats of both extract treatment and comparative control groups . culture extract on the biofilm formation of p. aeruginosa . since the genetic background of rats and the nature of biofilm influence the sensitivity of p. aeruginosa we are not able to conclude that the differences are only due to the effect of paenibacillus sp . the higher mortality seen in rat groups challenged with p. aeruginosa that were untreated with paenibacillus sp . culture extract compared to treated ones may be due to the ability of untreated isolates to produce higher amounts of elastase and other qs - regulated virulence factors . this was similar to the study by hoffmann et al . where they reported higher mortalities in mice challenged with the nonmucoid nh57388c isolate compared to the isogenic mucoid nh57388a isolate + qs . alginate from p. aeruginosa is a linear polymer of d - mannuronate and l - guluronate , which are highly soluble in water and chemically very similar to seaweed alginate . embedding p. aeruginosa with alginate solution enables the challenge strain to produce a more elastic biofilm structure as previously reported . culture extract caused weakness in the biofilm architecture and facilitated the clearance of its bacteria as visualized by both light microscope and confocal laser scanning microscope . histological examination of lung sections showed alveolar sacs invaded with p. aeruginosa biofilm anchored in a protective alginate matrix surrounded by numerous pmn . despite this significant pmn infiltration this finding supports previous in vitro studies showing that alginate protects bacteria from the host immune system . the lungs of rats were characterized by foci of p. aeruginosa biofilms in the alveoli and alveolar ducts ( figures 4(a)4(d ) ) . this was similar to the findings by tiddens where they have described the same pathological characteristics of those seen with cf patients . culture extract on the histology of lungs in both extract treatment and comparative groups were insignificant suggesting the same mechanism of action based on inhibiting the qs system of p. aeruginosa and thus preventing its persistence via the formation biofilm . aeruginosa lung infection in cf patients is characterized by a strong antibody response in the serum and infiltration of pmns in the lung . our results showed high serum levels of igm and igg among positive control group than that in both comparative and treatment groups . the increased levels of serum antibodies could lead to increased levels of immune complexes in the lung foci which are thought to play an important role in the immunopathology of cf . results of hematological profile and serum antibody responses revealed that the challenge p. aeruginosa inhibited the immune response of all experimental rats , except negative control group , during the early acute phase of infection characterized by a significant increase count of neutrophils , lymphocytes , and monocytes but stimulated the humoral immune reaction during the chronic infection characterized by the marked increase of igm , igg , and ige . our results indicates that qs systems significantly affected the severity of p. aeruginosa lung infection in both acute and chronic phases and that the qs signals such as ahl in gram - negative bacteria are promising new targets to combat virulence among patients with chronic p. aeruginosa lung infection . the activation of pmns and downregulation of the igg response might be associated with some changes in the production of cytokines . this was similar to the findings made by song et al . where they have reported the association of ginseng treatment on neutrophil chemiluminescence and immunoglobulin g subclasses in a rat model of chronic pseudomonas aeruginosa pneumonia . culture extract and its identified compounds in the regulation of cytokine response using an animal model of biofilm - associated infection . our study shows that culture extract of paenibacillus sp . can act as a potent antagonist of bacterial quorum sensing . application of the extract and its potential compounds to gram - negative biofilms , particularly p. aeruginosa , will increase bacterial susceptibility to antibiotics . natural products such as microbial extract have long been a source of medicines . however , most studies focus solely on bactericidal effects . since the microbial culture extract in this study showed little , bactericidal activity , qs remains a potential mode of action . a shift of our focus to anti - qs and antivirulence properties within the bacterial community may reveal new anti - qs compounds and provide a novel method for the treatment of bacterial infections . culture extract along with the 2(5h)-furanone control compound significantly increased the survival time of rats , assisted bacterial clearance , and reduced lung pathology , thus indicating that they could function as acyl - hsl antagonists and as quorum sensing inhibitors . attenuation of bacterial virulence rather than killing the pathogen might become a new concept for control of bacterial infections . this mode of action might not cause a selective pressure for the development of bacterial resistance to the antagonist . in a previous study by alasil et al . culture extract were identified using ultra performance liquid chromatography - diode array detection ( uplc - dad ) and liquid chromatography - mass spectrometry ( lc - ms ) . one of the reported compounds was a phospholipase a2 inhibitor with the name 4-hydroxy-5-(hydroxymethyl)-3-(14-methylpentadecanoyl ) tetronicacid-2(5h)-furanone with a similar chemical structure to the qs antagonist ( 5z)-4-bromo-5-(bromomethylene)-3-butyl-2(5h)-furanone from the algae delisea pulchra inhibiting biofilm formation in e. coli without inhibiting its growth . it is believed that the phospholipase a2 inhibitor compound in our paenibacillus sp . culture extract could interfere with the qs signal acyl - hsl that may influence the type of host immune responses and reduce the inflammation in vivo . however , more studies are needed to test the acute toxicity of this phospholipase a2 inhibitor and assess its therapeutic effects in vivo on reducing the biofilm - associated infections among a larger sample size . in conclusion , the effects of paenibacillus sp . culture extract on p. aeruginosa are complicated and extend beyond the domain of qs - control hypothesis . however , the inhibition of virulence factors and biofilm formation provide insights into how natural product ( metabolites ) derived from a novel bacterial species of paenibacillus can be used in the future to combat biofilm - associated infections caused by clinically important pathogens .
purposethe geometry of the tibial plateau and its influence on the biomechanics of the tibiofemoral joint has gained increased significance . however , no quantitative data are available regarding the inclination of the medial and lateral tibial slope in patients with patellar instability . it was therefore the purpose of this study to evaluate tibial slope characteristics in patients with patellar dislocations and to assess the biomechanical effect of medial - to - lateral tibial slope asymmetry on lateral patellar instability.methodsmedial and lateral tibial slope was measured on knee magnetic resonance images in 107 patients and in 83 controls . the medial - to - lateral tibial slope asymmetry was assessed as the intra - individual difference between the medial and lateral tibial plateau inclination considering severity of trochlear dysplasia . the effect of tibial slope asymmetry on femoral rotation was calculated by means of radian measure.resultsseverity of trochlear dysplasia was significantly associated with an asymmetric inclination of the tibial plateau . whereas the medial tibial slope showed identical values between controls and study patients ( n.s . ) , lateral tibial plateau inclination becomes flatter with increasing severity of trochlear dysplasia ( p < 0.01 ) . consequently , the intra - individual tibial slope asymmetry increased steadily ( p < 0.01 ) and increased internal femoral rotation in 20 and 90 of knee flexion angles in patients with severe trochlear dysplasia ( p < 0.01 ) . in addition , the extreme values of internal femoral rotation were more pronounced in patients with patellar instability , whereas the extreme values of external femoral rotation were more pronounced in control subjects ( p = 0.024).conclusiondata of this study indicate an association between tibial plateau configuration and internal femoral rotation in patients with lateral patellar instability and underlying trochlear dysplasia . thereby , medial - to - lateral tibial slope asymmetry increased internal femoral rotation during knee flexion and therefore might aggravate the effect of femoral antetorsion in patients with patellar instability.level of evidenceiii .	lateral patellar dislocation ( lpd ) is subject to a constant advancement of biomechanical understanding and treatment modalities . in recent years , it has become obvious that numerous risk factors have to be considered in clinical decision - making and that the understanding of an individual s anatomy is essential to identify who is at risk for subsequent instability episodes and to choose the most appropriate treatment . patellar stability is maintained by a complex interplay of active , passive and static stabilizers that act in harmony during knee motion . the static stabilizers are composed mainly of the joint geometry ; particularly , the shape of the trochlear groove prevents lpd , predominantly at ranges between 20 and 30 of knee flexion . concomitantly , abnormal joint geometry influences patellofemoral instability by means of different grades of trochlear dysplasia , by the anatomical and mechanical axis of the femur and tibia ( genu valgum ) , increased femoral antetorsion and external tibial rotation , patella height , and by an increased tibial tuberosity trochlear groove distance ( tt tg ) . in recent years , the geometry of the tibial plateau and its influence on the biomechanics of the tibiofemoral joint has gained increased significance . numerous investigators have evaluated the effect of tibial slope inclination on tibiofemoral contact area , joint translation , rotation and on the strain biomechanics of the cruciate ligaments [ 6 , 7 , 15 ] . in addition , knee anatomy , particularly the tibial slope , is directly associated with knee biomechanics exhibited during dynamic landings . although , the importance of the inclination of the tibial plateau is well established in the current literature , no quantitative data are available regarding the inclination of the medial and lateral tibial plateau and its influence on the biomechanics of the tibiofemoral joint in patients with lpd . therefore , it was the objective of this study to evaluate the geometry of the tibial plateau in patients with lpd . specifically , we addressed the following research questions : ( a ) is lateral patellar instability associated with a modified inclination of the medial and lateral tibial plateau ( tibial slope ) and , if so , ( b ) in what way does this modification influence tibiofemoral biomechanics ? it was the hypothesis of the current study that patients with patellofemoral instability show also significant changes in the anatomy and the biomechanics of the tibiofemoral joint . the study group was composed of 107 patients ( male / female , 55/52 ; age range 1055 years ) with 107 knee magnetic resonance imaging ( mri ) investigations who had been treated for lpd between february 2006 and june 2010 . a picture archiving and communications system ( pacs ) workstation ( centricity , ge healthcare , st . gilles , united kingdom ) was used to identify patients with acute or recurrent patellar dislocations . the criteria for lpd included joint effusion , contusion on the lateral femoral condyle or medial patella margin , osteochondral fragments , injury to the medial ligamentous stabilizers and a lateralized patella . the mri - based diagnosis of lpd was made in patients who met three or more of the criteria . additionally , to be included patients had to have a convincing history of primary or recurrent patellar dislocations with the clinical symptom of giving way and the clinical findings of joint effusion , tenderness along the medial retinaculum , medial patella or medial femoral condyle , and a positive apprehension sign . the criteria for exclusion were any proximal or distal realignment procedures , trochleoplasty , fractures of the distal femur or tibial head , and a multiligament - injured knee joint . these criteria were used to identify 107 patients , who formed the study group for this investigation . knee mri investigations of 83 patients ( male / female , 42/41 ; age range 1259 years ) served as controls . investigations were performed during the same time period , and patients were matched for age and gender . investigations were performed due to an internal derangement of the knee such as meniscus tear or cartilage lesion of the tibiofemoral joint . in addition , patients with an anterior cruciate ligament ( acl ) tear were also excluded because this injury is often associated with an increased inclination of the tibial plateau in non - contact acl injury mechanisms . in all patients coronal , sagittal and transverse mri images were available and were used to measure the medial and lateral tibial slope , the anatomical distal lateral femur angle ( adlfa ) , the mechanical proximal medial tibial angle ( mpmta ) , and the degree of trochlear dysplasia . measurements were performed with the use of the annotation tools on the digital pacs workstation . the software presented the angular values and length measurements to one decimal place that were rounded into a single - digit integer format . this means , if the true value of an angle was , for example , 5.4 , this was rounded off to 5. on the other hand , if the actual angle was 5.5 , this was rounded up to 6. similarly , if the measured length was 40.5 mm , this was rounded up to 41 mm , and if the actual length was 40.4 mm , this was rounded off to 40 mm . therefore , all measurements were made with a sensitivity of 1 ( 0.5 to 0.5 ) and 1 mm ( 0.5 to 0.5 mm ) , respectively . to answer the first research question , the medial and lateral tibial slope was assessed according to the protocol proposed by hashemi et al . . initially , the first proximal slice on transverse mr images that showed the entire tibial head was identified ( fig . 1 ) . in this transverse image , the corresponding sagittal slice located most closely to the tibial head centre was determined ( solid line in fig . 1 ) . the longitudinal axis was defined as the midpoint of the anterior posterior width of the tibia at two points located 45 cm distally to the joint line and as distally as possible . the conjugation line of these two points represents the longitudinal axis of the tibia in the sagittal plane . to measure the medial and lateral tibial slope , the longitudinal axis was assigned to the corresponding planes in the centre of the medial tibial plateau and in the centre of the lateral tibia plateau , shown as dashed lines in fig . 1 . again , the corresponding planes were reproduced in the medial and lateral sagittal image ( fig . the conjugation line between the peak anterior and posterior points of the bony tibia plateau measured the inclination of the tibial slope perpendicular to the longitudinal axis of the tibia . the tibial slope was defined as positive if the peak anterior point lay above the peak posterior point and was defined as negative if the posterior point lay above the anterior point.fig . 1first transverse mr image in craniocaudal direction that shows the entire tibial head . in this transverse image , the corresponding sagittal slices located most closely to the tibial head centre ( solid line ) and in the centre of the medial and lateral tibial plateau ( dashed lines ) were determined . similarly , the corresponding coronal section located most closely to the tibial head centre ( dotted line ) was establishedfig . 2the sagittal plane that represents the corresponding image to the solid line in fig . the longitudinal axis was defined as the midpoint of the anterior posterior width of the tibia at two points located 45 cm distally to the joint line and as distally as possible . to measure the medial and lateral tibial slope , the longitudinal axis was assigned to the corresponding planes in the centre of the lateral tibial plateau ( b ) and in the centre of the medial tibia plateau ( c ) as shown as dashed lines in fig the conjugation line between the peak anterior and posterior points of the tibia plateau measured the inclination of the tibial slope perpendicular to the longitudinal axis of the tibia first transverse mr image in craniocaudal direction that shows the entire tibial head . in this transverse image , the corresponding sagittal slices located most closely to the tibial head centre ( solid line ) and in the centre of the medial and lateral tibial plateau ( dashed lines ) were determined . similarly , the corresponding coronal section located most closely to the tibial head centre ( dotted line ) was established the sagittal plane that represents the corresponding image to the solid line in fig . the longitudinal axis was defined as the midpoint of the anterior posterior width of the tibia at two points located 45 cm distally to the joint line and as distally as possible . to measure the medial and lateral tibial slope , the longitudinal axis was assigned to the corresponding planes in the centre of the lateral tibial plateau ( b ) and in the centre of the medial tibia plateau ( c ) as shown as dashed lines in fig . 1 . the conjugation line between the peak anterior and posterior points of the tibia plateau measured the inclination of the tibial slope perpendicular to the longitudinal axis of the tibia a comparable method was used to measure the adlfa and mpmta , according to paley et al . first , the longitudinal axis of the tibia in the frontal plane was established referencing most closely to the centre of the tibial head in the corresponding transverse image ( dotted line in fig . 1 ) . again , the longitudinal axis was defined as the midpoint of the medial - to - lateral width of the tibia as distally as possible and at the midpoint of the tibial head . a similar approach was used to establish the diaphyseal axis of the femur in the coronal plane . the adlfa and mpmta were then measured as the angle between the longitudinal axis of the femur and tibia and the joint line represented by the most distally located points of the femoral condyles , and the peak points of the medial and lateral tibial plateau , respectively ( fig . 3the longitudinal axis of the femur and the tibia was established in the frontal plane referencing most closely to the centre of the tibial head in the corresponding transverse image ( dotted line in fig . 1 ) . the longitudinal axis was defined as the midpoint of the medial - to - lateral width of the tibia as distally as possible and at the midpoint of the tibial head . a similar approach was used to establish the diaphyseal axis of the femur in the coronal plane . the adlfa and mpmta was then measured as the angle between the longitudinal axis of the femur and tibia and the joint line represented by the most distally located points of the femoral condyles , and the peak points of the medial and lateral tibial plateau . adlfa anatomical distal lateral femur angle , mpmta mechanical proximal medial tibial angle the longitudinal axis of the femur and the tibia was established in the frontal plane referencing most closely to the centre of the tibial head in the corresponding transverse image ( dotted line in fig . 1 ) . the longitudinal axis was defined as the midpoint of the medial - to - lateral width of the tibia as distally as possible and at the midpoint of the tibial head . a similar approach was used to establish the diaphyseal axis of the femur in the coronal plane . the adlfa and mpmta was then measured as the angle between the longitudinal axis of the femur and tibia and the joint line represented by the most distally located points of the femoral condyles , and the peak points of the medial and lateral tibial plateau . adlfa anatomical distal lateral femur angle , mpmta mechanical proximal medial tibial angle trochlear dysplasia was assessed on the transverse mr images as described by fucentese et al . . : type a : trochlear morphology preserved with a fairly shallow trochlea ; type b : flat or convex trochlea ; type c : asymmetry of trochlear facets with convex lateral facet and hypoplastic medial facet ; and type d : asymmetry of trochlear facets , hypoplastic medial facet and cliff pattern . to answer the second research question , the analyses by ngerl et al . and pinskerova et al . these studies showed that the contact areas of the femoral condyles on the tibial plateau move backwards during knee flexion . under weight - bearing conditions , the contact point between the medial femoral condyle and the tibial plateau averaged a distance of 29 and 22 mm measured from the ipsilateral posterior tibial cortex in 20 and 90 of knee flexion , respectively . this means that a difference of the medial and lateral tibial plateau inclination would influence femoral rotation by means of a different height between the posteromedial and posterolateral femorotibial contact points as illustrated in fig . 4a and b. thus , the medial - to - lateral tibial slope asymmetry was assessed as the intra - individual difference between the medial and lateral tibial plateau inclination . this means that the tibial slope asymmetry had a positive sign in cases where the medial slope was steeper than the lateral slope , and had a negative sign in cases where the medial slope was flatter than the lateral slope.fig . 4illustrated is the effect of tibial slope asymmetry on femoral rotation by means of a difference in height between the medial and lateral tibial plateau in 20 and 90 of knee flexion . in the frontal view , the maximal observed effect on internal and on external femoral rotation is shown in comparison with the neutral position of the femoral condyles in both knee flexion angles illustrated is the effect of tibial slope asymmetry on femoral rotation by means of a difference in height between the medial and lateral tibial plateau in 20 and 90 of knee flexion . in the frontal view , the maximal observed effect on internal and on external femoral rotation is shown in comparison with the neutral position of the femoral condyles in both knee flexion angles the effect of tibial slope asymmetry on femoral rotation was calculated mathematically by means of radian measure with the knee positioned in 20 and 90 of knee flexion . first , the value of tibial slope asymmetry ( in degrees ) was converted into radian according to the formula rad = deg /180 . this value was then multiplied either by 23 mm or by 30 mm , representing the difference in height ( in mm ) between the posteromedial and posterolateral contact points in 20 or 90 knee flexion . the value of 23 and 30 mm resulted from the length of the tibial head in the sagittal plane ( 52 mm ; n = 35 ) minus the distance from the posterior tibial cortex to the medial femorotibial contact point ( 29 mm for 20 of knee flexion ; 22 mm for 90 of knee flexion ) , according to pinskerova et al . . subsequently , the differences in height were divided by the distance between the centre of the medial and lateral femoral condyle in the frontal plane ( intercondylar distance : 44 mm ; n = 35 ) representing the value of femoral rotation in radian . values of radian measures were then converted into degrees ( deg = rad 180/ ) representing internal femoral rotation in cases of positive values and external femoral rotation in cases of negative values . the influence of the grade of trochlear dysplasia onto the medial and lateral tibial slope and onto age , adlfa , and mpmta was individually assessed by linear regression with trochlear dysplasia as independent variable . since age was significantly associated with the grade of trochlear dysplasia , it was added as an independent variable into the other regression models . gender and lpd were compared between the different types of trochlear dysplasia using the -exact test . the portion of study patients and controls on the 20 largest slope asymmetries , in positive and in negative direction , respectively , was evaluated by fisher s exact test . because lpd and trochlear dysplasia were nearly perfectly correlated , an adjustment for age was not possible for this analysis . to study intrarater and interrater reliability , two measurement series on 20 randomly taken mri were drawn either repeatedly by 1 single rater with a 2-week interval or independently by 2 different raters . reliability was assessed by the correlation ( pearson r ) between the two measurement series or by the mean difference ( t test ) between them . all analyses were performed using the free software r ( version 2.12 , www.r-project.org ) . the testing level was chosen to be alpha = 5 % for all tests . the influence of the grade of trochlear dysplasia onto the medial and lateral tibial slope and onto age , adlfa , and mpmta was individually assessed by linear regression with trochlear dysplasia as independent variable . since age was significantly associated with the grade of trochlear dysplasia , it was added as an independent variable into the other regression models . gender and lpd were compared between the different types of trochlear dysplasia using the -exact test . the portion of study patients and controls on the 20 largest slope asymmetries , in positive and in negative direction , respectively , was evaluated by fisher s exact test . because lpd and trochlear dysplasia were nearly perfectly correlated , an adjustment for age was not possible for this analysis . to study intrarater and interrater reliability , two measurement series on 20 randomly taken mri were drawn either repeatedly by 1 single rater with a 2-week interval or independently by 2 different raters . reliability was assessed by the correlation ( pearson r ) between the two measurement series or by the mean difference ( t test ) between them . all analyses were performed using the free software r ( version 2.12 , www.r-project.org ) . the testing level was chosen to be alpha = 5 % for all tests . severity of trochlear dysplasia was significantly associated with an asymmetric inclination of the tibial plateau ( table 1 ) . whereas the medial tibial slope showed identical values between controls and study patients ( n.s . ) , lateral tibial plateau inclination becomes flatter with increasing severity of trochlear dysplasia ( p < 0.01 ) ( table 1 ; fig . 5 ) . consequently , the intra - individual slope asymmetry increased steadily towards positive values ( p < 0.01 ) and therefore increased internal femoral rotation in 20 and in 90 of knee flexion in patients with severe trochlear dysplasia ( p < 0.01 ) ( table 1 ; fig . 6 ) . though tibial slope asymmetry showed a relatively wide range in both study and control patients , study patients were more frequently found than controls among the 20 largest positive values of tibial slope asymmetry ( 610 ) . on the other hand , among the 20 largest negative values of tibial slope asymmetry ( 4 to 8 ) , control patients were more often found than study patients ( p = 0.024 ) . concomitantly , this means that the extreme values of internal femoral rotation were more pronounced in patients with lpd , whereas the extreme values of external femoral rotation were more pronounced in control subjects . the study group also showed a slightly increased valgus deformity expressed by a smaller adlfa ( p < 0.01 ) , whereas the mpmta did not differ between study and control subjects ( n.s . ) . interrater and intrarater reliability was near - perfect for the investigated parameters with a clearly positively significant correlation between the two measurement series ( table 2a , b).table 1distribution of patient characteristics and study parameters considering different grades of trochlear dysplasiaparametergrade of trochlear dysplasia p valuenormal ( n = 90)type a ( n = 18)type b ( n = 47)type c ( n = 23)type d ( n = 12)age ( years)27.2 9.6(12.059.0)22.6 7.1(13.038.0)24.9 9.7(10.055.0)20.5 5.3(13.030.0)18.0 6.7(12.038.0)<0.01gendern.s . male48 ( 53 % ) 9 ( 50 % ) 26 ( 55 % ) 10 ( 43 % ) 5 ( 42 % ) female42 ( 47 % ) 9 ( 50 % ) 21 ( 45 % ) 13 ( 47 % ) 7 ( 58 % ) dislocation<0.01 yes7 ( 8 % ) 18 ( 100 % ) 47 ( 100 % ) 23 ( 100 % ) 12 ( 100 % ) no83 ( 92 % ) 0 ( 0 % ) 0 ( 0 % ) 0 ( 0 % ) 0 ( 0 % ) tibial slope medial7.0 3.9(1.0 to 15.0)7.3 4.5(0.014.0)7.6 4.3(1.5 to 20.0)6.8 3.8(0.5 to 14.0)6.5 4.5(1.017.0)n.s.tibial slope lateral6.7 3.9(0.5 to 15.5)7.1 5.3(0.015.5)6.1 4.8(0.018.0)4.5 4.6(5.0 to 14.5)3.0 3.4(1.0 to 9.5)<0.01slope asymmetry ( )0.2 3.8(8.0 to 7.0)0.2 2.8(4.5 to 5.0)1.5 3.4(8.0 to 9.0)2.3 3.8(4.0 to 10.0)3.5 3.4(1.5 to 8.0)<0.01femoral rotation in 20 knee flexion0.1 1.9(4.2 to 3.7)0.1 1.4(2.4 to 2.6)0.8 1.8(4.2 to 4.7)1.2 2.1(2.3 to 5.5)1.9 1.8(0.7 to 4.2)<0.01femoral rotation in 90 knee flexion0.1 2.6(5.5 to 4.8)0.2 1.9(3.0 to 3.4)1.0 2.3(5.5 to 6.1)1.6 2.7(3.0 to 7.2)2.5 2.4(1.0 to 5.5)<0.01adlfa ( )82.8 2.2(76.088.0)81.3 2.4(76.586.0)81.6 2.4(77.086.0)81.0 3.0(76.086.5)81.5 2.4(77.085.5)<0.01mpmta ( )86.1 2.5(80.591.0)85.3 2.1(82.088.5)85.8 2.2(82.090.0)86.0 2.0(82.090.0)86.4 2.1(83.089.0)n.s.*descriptive values are presented as either absolute ( relative ) frequencies or mean standard deviation ( minimum maximum ) . not significant , adlfa anatomical distal lateral femur angle , mpmta mechanical proximal medial tibial anglefig . 5shown are the mean values and sd of the medial and the lateral tibial slope in controls and in patients considering different grades of trochlear dysplasiafig . 6shown are the mean values and sd of internal femoral rotation in controls and in patients considering different grades of trochlear dysplasia with the knee positioned in 20 and 90 of knee flexiontable 2correlation and mean of differences between 2 measurement series on the same 20 individuals , ( a ) drawn repeatedly by 1 single rater , ( b ) 2 different raterspearson r p valuemean of differences p valuea intrarater reliability medial slope0.92<0.010.4n.s . not significant distribution of patient characteristics and study parameters considering different grades of trochlear dysplasia descriptive values are presented as either absolute ( relative ) frequencies or mean standard deviation ( minimum maximum ) . not significant , adlfa anatomical distal lateral femur angle , mpmta mechanical proximal medial tibial angle shown are the mean values and sd of the medial and the lateral tibial slope in controls and in patients considering different grades of trochlear dysplasia shown are the mean values and sd of internal femoral rotation in controls and in patients considering different grades of trochlear dysplasia with the knee positioned in 20 and 90 of knee flexion correlation and mean of differences between 2 measurement series on the same 20 individuals , ( a ) drawn repeatedly by 1 single rater , ( b ) 2 different raters n.s the most important finding of this study was that the individual tibial slope asymmetry , expressed as the difference between the medial and the lateral tibial slope , increased linearly considering severity of trochlear dysplasia . this increase in tibial slope asymmetry was based on a decrease in the lateral tibial slope with consistent inclination of the medial slope . tibial slope asymmetry therefore influenced femoral rotation by means of a different height between the posterior medial and posterior lateral tibial head . thereby , internal femoral rotation was more pronounced in patients with lpd , whereas external femoral rotation was more pronounced in control subjects without patellofemoral instability . links between knee joint biomechanics and joint morphology have been well described in recent years [ 1 , 10 ] . particularly , the effect of the geometry of the tibial plateau on the strain biomechanics of the cruciate ligaments has gained increased significance . thereby , the combination of steep medial and lateral tibial slopes with a shallow concavity of the medial tibial head increased the risk of non - contact acl injuries . moreover , the geometry of the tibial slope also influenced the peak stance knee internal rotation during dynamic landings . specifically , when the lateral tibial slope dominated tibial plateau geometry , the peak internal rotation angle was increased , indicating that the geometry of the tibial plateau exerts a dynamic role on knee rotational alignment . similarly , our results support an association between tibial plateau configuration and internal femoral rotation in patients with lateral patellar instability and underlying trochlear dysplasia . thereby , medial - to - lateral tibial slope asymmetry increased internal femoral rotation during knee flexion , potentially aggravating the effect of femoral antetorsion in patients with lpd . in addition , it seems plausible that tibial slope asymmetry also changes the knee axial rotation response . with the longitudinal tibial rotation axis located on the medial tibial plateau , a relative increase in the medial slope will shift the medial tibiofemoral contact point anteriorly . concomitantly , a relative shallow lateral slope will reduce the anterior directed tibiofemoral translation force on the lateral side , culminating in greater external tibial rotation . thus , positive values of tibial slope asymmetry might influence patellofemoral instability by both , an increase in internal femoral rotation and an increase in external tibial rotation . one has to consider that a smaller radius of curvature of the lateral femoral condyle might reduce the effect on internal femoral rotation . three distinct features the femoral bicondylar angle , the prominence of the lateral lip of the femoral trochlear , and the elliptic profile of the lateral condyle characterize the development of the trochlear groove and the distal femur . the increase in bicondylar angle is correlated with the disproportionate anteroposterior development of the lateral pillar . we therefore measured the radius curvature of 35 study patients ( trochlear dysplasia type c and d ) and of 35 at random selected patients of the control group . no significant difference was found between both groups ( data not shown ) so that the lateral condyles radius of curvature seems unlikely to compensate for the asymmetric tibial slope effect . recently , hashemi et al . have already pointed out that the true tibial slope should be measured at the centre of the articular regions of the medial and lateral tibial plateau . they found a relatively wide range for both the medial and the lateral slope with ranges between 3 and 10 and between 0 and 14 , respectively . similarly , matsuda et al . reported medial and lateral tibial slope ranges between 5 and 15.5 and between 0 and 14.5 , respectively . we also found a high range within measurements of medial ( 1.5 to 20 ) and lateral tibial slope ( 5 to 18 ) , moderately exceeding those of previous studies . however , this difference might be attributable to a much higher number of subjects included in our study ( n = 190 ) when compared to the analysis by hashemi et al . ( n = 60 ) [ 5 , 8 ] . to the best of our knowledge , this is the first study that investigated the association between lpd and tibial plateau geometry . in addition , this study aims to provide an initial understanding of medial - to - lateral tibial slope asymmetry and the resultant biomechanical characteristics of the tibiofemoral joint with its relevant contributions to lateral patellar instability . if one considers the mean values of tibial slope asymmetry and femoral rotation , it could be argued that they may not have a dramatic effect on the biomechanics of the knee joint . however , the important point to consider is the differences between the extreme values of internal femoral rotation ( 5.5 in 20 knee flexion ; 7.2 in 90 knee flexion ) in patients with patellar instability and that of external femoral rotation ( 4.2 in 20 knee flexion ; 5.5 in 90 knee flexion ) in control subjects . first , as already stated by hashemi et al . , it is important to note that the access to a sufficient length of the femur and tibia in the magnetic resonance image and the ability to identify landmarks , precisely all , could have an impact on the slope measurements . second , slope measurements were referenced on the bony landmarks in the centre of the medial and lateral tibial plateau , but knee joint morphology is further characterized by the cartilage surface that is concave medially and convex laterally . this means that the articular surface is higher on the lateral side and lower on the medial side when compared to the bony landmarks used in this study . this in turn , however , would enhance the observed effect on internal femoral rotation during knee flexion rather than diminish it . finally , our analyses were based on an idealized mathematical model with the knee positioned in 20 and 90 of knee flexion . it was not able to evaluate the effect of tibial slope asymmetry on tibiofemoral biomechanics during varying degrees of knee flexion . if one considers that the patella becomes least stable between 0 to 30 of knee flexion , it could be argued that an increase in internal femoral rotation may not have a dramatic effect on patellar instability in higher degrees of knee flexion . however , though most patellae dislocate from a nearly straight start followed by a movement to flexion , there is a certain amount of patients dislocating their patellae from a well - bent start followed by either knee extension or flexion . thus , it seems likely that an increase in internal femoral rotation contributes relevantly to patellar instability even in higher degrees of knee flexion . knowledge of the influence of medial - to - lateral tibia slope asymmetry on femoral rotation might help physicians in the decision - making process towards a torsional osteotomy in patients with lateral patellar instability and a marginally increased femur antetorsion . the present study introduces a new aspect in the complex interplay between the patellofemoral and the tibiofemoral joint considering the biomechanics of lateral patellar instability . it shows that the individual tibial slope asymmetry , expressed as the difference between the medial and the lateral tibial slope , increased linearly considering the severity of trochlear dysplasia and , concomitantly , influenced femoral rotation during knee flexion . thereby , internal femoral rotation was more pronounced in patients with lpd , whereas external femoral rotation was more pronounced in control subjects without patellofemoral instability . this article is distributed under the terms of the creative commons attribution license which permits any use , distribution , and reproduction in any medium , provided the original author(s ) and the source are credited .
memory consolidation requires transcription and translation of new protein . arc , an effector immediate early gene , and zif268 , a regulatory transcription factor , have been implicated in synaptic plasticity underlying learning and memory . this study explored the temporal expression profiles of these proteins in the rat hippocampus following fear conditioning . we observed a time - dependent increase of arc protein in the dorsal hippocampus 30-to-90-minute post training , returning to basal levels at 4 h. zif268 protein levels , however , gradually increased at 30-minute post training before peaking in expression at 60 minute . the timing of hippocampal arc and zif268 expression coincides with the critical period for protein synthesis - dependent memory consolidation following fear conditioning . however , the expression of arc protein appears to be driven by context exploration , whereas , zif268 expression may be more specifically related to associative learning . these findings suggest that altered arc and zif268 expression are related to neural plasticity during the formation of fear memory .	a predominant question in neuroscience is how memory functions are supported by the central nervous system and what cellular processes are necessary . one focus of this research is on protein - dependent synaptic modifications that occur as a consequence of neuronal activity . signaling cascades activated at the time of learning can induce the transcription of particular genes , ultimately leading to de novo protein synthesis and subsequent structural changes to support long - term memories . immediate - early genes ( iegs ) are induced soon after neuronal activity , and they participate in diverse functions . some iegs are regulatory transcription factors ( e.g. , zif268/egr1 ) responsible for inducing transcription of late - response genes , while others are effector iegs ( e.g. , arc / arg3.1 ) that are directly involved in cellular changes at locations such as the cytoskeleton or receptors . however , the transcripts of some iegs , such as activity - regulated cytoskeleton - associated protein ( arc ) , are transported to the dendrites and protein synthesis occurs there , thus making arc a reasonable target for researchers investigating the underlying mechanisms of postsynaptic changes supporting memory formation . arc ( also called arg3.1 ) is a plasticity - related gene whose induction occurs soon after synaptic activation [ 24 ] , mrna transcription is independent of de novo protein synthesis , and expression is primarily in excitatory neurons following behavioral experience . arc contains a synaptic activity - responsive element ( sare ) in the promoter upstream of the initiation site , which is necessary for transcription and sufficient for the induction of activity - dependent arc . arc mrna is transported to the dendrites [ 3 , 4 , 6 ] perhaps via sumoylation ( reviewed in ) , where it is intradendritically localized to activated synapses by phosphorylated erk ( extracellular signal - regulated kinase ) signaling and actin polymerization [ 6 , 811 ] , translated into protein , and becomes a part of the postsynaptic junction . the recruitment of arc to the dendrites suggests its importance for synaptic plasticity that occurs after activation . arc expression has been strongly linked to long - term potentiation ( ltp ) and learning . high frequency stimulation ( hfs ) induces both ltp and arc expression , which are dependent upon nmda receptor activation [ 3 , 4 ] but not upon the activation of ampa receptors . additionally , intrahippocampal infusions of arc antisense in vivo disrupt multiple aspects of ltp , indicating that arc protein synthesis is required for the early expression , maintenance , and consolidation of enduring ltp ( [ 13 , 14 ] , reviewed in ) . in accordance with ltp as a molecular model for learning and memory , delivery of arc antisense to the dorsal hippocampus produces long - term memory deficits in spatial water maze performance and inhibitory avoidance in rats , indicating a necessary role for arc protein in memory consolidation . furthermore , arc - knockout mice show impaired spatial learning in the morris water maze task , disrupted fear memory to context and auditory stimuli , and deficits in conditioned taste aversion and object recognition . recent findings provide evidence for the role of arc in the regulation of ampa receptors through interactions with endocytic proteins in dendrites ( [ 17 , 18 ] , reviewed in [ 19 , 20 ] ) , as well as a function in the stabilization and the expansion of the f - actin cytoskeleton at activated synapses , strengthening the argument that arc is involved in modifications that affect synaptic efficacy ( reviewed in ) . the protein product of the immediate early gene zif268 ( also termed egr1 , or early growth response gene ) is a transcription factor of the zinc finger family . expression of zif268 is regulated by synaptic activity and dependent upon nmda receptor activation . induction of ltp produces increased expression of zif268 mrna , and knockout of the zif268 gene in mice results in absent late ltp in the hippocampus and deficits in long - term memory for spatial water maze , conditioned taste aversion , socially transmitted food preference , and object recognition . additionally , infusions of zif268 antisense into the amygdala prior to contextual fear conditioning disrupt fear memory consolidation . in the present set of experiments , we used pavlovian fear conditioning to investigate the time - dependent expression of arc and zif268 . in pavlovian fear conditioning , a neutral stimulus is paired with an aversive unconditional stimulus ( ucs ) . through this pairing , the once neutral stimulus becomes able to elicit a fear response ( termed the conditional response , or cr ) . the animal also acquires fear for the context in which fear conditioning occurred . when the animal is presented with the shock - associated auditory stimulus or is placed back in the training context , it will exhibit fear behaviors indicating memory for the training experience . the amygdala is crucial for the acquisition , consolidation , and expression of classically conditioned fear , as it receives information about both conditional and unconditional stimuli ( cs and ucs , respectively ) making it a site of associative convergence [ 25 , 26 ] . amygdala lesions and protein - synthesis inhibitors delivered to the amygdala disrupt fear conditioning [ 2729 ] . the hippocampus is not necessary for conditional fear to an auditory cs in a delay paradigm , but it is essential for contextual fear . post training hippocampal lesions abolish contextual fear in rodents , but they do not affect conditioning to an auditory stimulus and protein - synthesis inhibitors given into the hippocampus block the acquisition of contextual fear memory [ 26 , 31 ] . the present study examined the time course of arc protein expression in the hippocampus following pavlovian fear conditioning . in addition , the temporal profile of zif268 , another plasticity - related gene product , was measured and compared to the pattern of expression for arc protein . immunohistochemistry followed western blot studies to show the localization of arc and zif268 in hippocampal regions with elevated protein expression post training . additional control groups for shock stimulation and simple exposure to auditory and contextual stimuli were analyzed with western blots to better determine the specific contribution of arc and zif268 protein in the hippocampus . in all experiments , male long evans rats ( n = 148 ; harlan ; madison , wi ) weighing approximately 350 g were used . the animal colony was climate - controlled and maintained on a 14 hr : 10 hr light : dark cycle with lights on at 7:00 a.m. all experimental procedures were performed during the light cycle . all procedures were approved by the institutional animal care and use committee at the university of wisconsin - milwaukee . each chamber was constructed from clear plexiglas ( front and back walls , ceiling ) and stainless steel ( side walls ) and measured 28 20.5 21 cm ( length height depth ) . stainless steel rods spaced 12 mm apart served as the floor of the chamber and were used to deliver a mild footshock from a scrambled shock generator . conditioning chambers were housed in sound - attenuating boxes that were illuminated by a 7.5 w white light bulb . there was a constant background noise of 5660 db produced by ventilation fans inside the boxes . the chambers were cleaned with 5% ammonium hydroxide solution between each rat . in all behavioral testing , the dependent measure was freezing behavior , which is operationally defined as the lack of all movement , except movement necessary for respiration . the training procedure was recorded by video cameras installed inside the sound - attenuating chambers , and freezing behavior was scored by computer software ( freezescan 1.0 ; clever sys . , rats were adapted to handling and transportation procedures for 3 min each on 6 consecutive days . rats were trained in a single 15-min session of auditory - cued fear conditioning ( figure 1(a ) ) . after an initial 6-min baseline period , the rats received four presentations of white noise ( 72 db , 10 s ) that coterminated with a footshock ( 1.3 ma , 1 s ) . the rats remained in the chamber for an additional 4 min following the last footshock before being returned to their home cages . this training protocol has previously been shown to produce both contextual and auditory - cued fear memories [ 28 , 29 , 33 ] . additional groups of animals were created to control separately for auditory and contextual experience and shock stimulation . one group experienced the same training protocol but with no shock stimuli delivered ( wn - cxt ) , and another group received footshock immediately upon placement in the chamber and removed shortly afterward ( shk - only ) . after training , animals were returned to their home cages and later euthanized with an overdose intraperitoneal injection of a sodium pentobarbital solution at varying time points post training . animals were killed at 30 min , 60 min , 90 min , 4 hr , 8 hr , 12 hr , or 24 hr after training , and nave home cage ( hc ) rats served as a control group . in a separate experiment , wn - cxt and shk - only groups were euthanized 60 min after stimulus exposure and were compared to trained rats also killed 60-min post training as well as additional hc controls . it was not possible to control the animals ' behavior ( e.g. , sleeping ) during the survival interval , however , the occurrence of any such behaviors prior to euthanasia should be controlled for by the hc group . hc animals were killed at varying times during the day across the experiment to account for circadian patterns , unsystematic animal behavior , and any variation in the animal colony . thus , the protein expression measured in the trained groups beyond that observed in the hc animals should be specific to the learning experience and not the result of unsystematic variability . euthanized rats were decapitated and the brains were quickly removed , frozen on dry ice , and stored at 80c . tissue samples were microdissected from the dorsal hippocampus ( figure 1(b ) ) . in all dissections , a rat brain atlas and a rat brain matrix ( harvard apparatus , holliston , ma ) were used to maintain consistency in tissue collection . hippocampal samples were homogenized manually with a pestle and glass tissue grinder in a buffer solution ( all in 100 ml ddh2o : 0.605 g tris - hcl , 0.25 g sodium deoxycholate , 0.876 g nacl , 0.038 g edta , 0.0042 g naf , 1 g / ml pmsf , 1 g / ml leupeptin , 1 g / ml aprotinin , 10 ml 10% sds , 1 mm sodium orthovanadate ) until there were no visible traces of solid matter . homogenates were stored in centrifuge tubes and kept frozen at 80c until time for centrifugation ( 4000 rpm for 20 min ) . the supernatant was removed , placed into small centrifuge tubes , and stored at 80c . a bradford protein assay was performed to determine the total amount of protein in the samples ( bio - rad dc protein assay kit , hercules , ca ) . sample dilutions were pipetted into 96-well plates and compared to serial dilutions of the protein standard ( bio - rad bsa 1.35 mg / ml ) using a versamax plate reader and softmax pro 4.3 ls software . samples were discarded from further analysis if they did not meet a set criterion for variance , and this standard was applied to all groups equivalently . normalized protein samples were loaded onto 7.5% sds gels for arc blots or 9.0% sds gels for zif268 blots using a mini protean holder filled with electrophoresis running buffer and a bio - rad powerpac ( 200 v , 0.04 a constant , 90 min ) . each experimental condition was represented on each gel to counterbalance any slight variation in blot development . gels were washed in transfer buffer ( 3.03 g tris , 14.4 g glycine , 200 ml methanol , 5 ml 10% sds , ddh2o up to 1 l ) before the protein was transferred to pvdf membranes using a semidry transfer cell ( bio - rad ; powerpac settings : 15 v constant , 2.00 a , 75 min ) . after protein transfer , membranes were incubated for 2 hr in blocking buffer ( 500 ml tbs , 15 g nonfat dry milk ) and then exposed to primary antibody for 90120 min . a monoclonal antibody for arc protein ( dilution 1 : 100 in antibody buffer , santa cruz ) , a polyclonal antibody for zif268 protein ( dilution 1 : 1000 , cell signaling ) , and a polyclonal antibody for -actin ( dilution 1 : 1000 , cell signaling ) were used in these experiments . after exposure to the primary antibody , membranes were washed twice for 15 min in antibody buffer ( 100 ml blocking buffer , 50 l tween-20 ) before being incubated for 90120 min in secondary antibody ( goat anti - mouse for arc blots1 : 5000 dilution , santa cruz ; goat anti - rabbit for zif268 and -actin blots1 : 2000 dilution , upstate biotechnology ) . membranes were washed twice for 15 min in wash buffer ( 100 ml tbs , 50 l tween-20 ) before exposure to chemiluminescence solution ( santa cruz ) for 3 min . washes and incubations were generally done at room temperature , however , primary incubation was sometimes performed overnight at 4c and then for 1 hr at room temperature . developments were conducted in a dark room , where membranes with chemiluminescence were exposed to autoradiographic film in a cassette . any disruptions in the signal during development of the films caused the sample to be excluded from further analysis . the bands representing arc ( molecular weight ( mw ) : 55 kda ) , zif268 ( mw : 75 kda ) , and -actin ( mw : 45 kda ) were measured using densitometry software ( nih imagej ) . optical density measures were computed for each hippocampal sample as a percentage of the home - cage animals ' ( control group ) protein expression , and these results were statistically analyzed using one - way anovas and fisher 's least significant difference ( lsd ) post - hoc comparisons when appropriate . rats ( n = 6 ) were euthanized with an overdose of isofluorane either 30 or 90 min after a single session of fear conditioning ( 15-min session with signaled shocks , see figure 1(a ) ) . the 30-min and 90-min time points were selected based on the time course for zif268 and arc expression established by the western blot analysis . the rats were perfused transcardially with 0.1 m pbs followed by 10% buffered formaldehyde prior to decapitation . brains were removed and placed in 10% buffered formaldehyde overnight , and then transferred to 30% sucrose formalin for cryoprotection for another 24 hr . prior to slice collection , brains were rinsed 3 times in 0.1 m pbs for 10 min each . using a freezing microtome , coronal slices ( 50-micron thick ) were collected throughout the rostral - caudal extent of the dorsal hippocampus and placed into 24-well plates with 500 l of 0.1 m pbs . the slices were incubated on a titer plate in 1% sodium borohydride for 15 min , 0.1 m pbs twice for 10 min each , 10% normal goat serum for 30 min , and primary antibody for 30 min . arc ( 1 : 100 dilution ) , zif268 ( 1 : 500 dilution ) , and neun ( 1 : 200 dilution ) primary antibodies were used to determine the regional localization and neuronal colocalization of arc and zif268 proteins . neun antibody ( chemicon - millipore ) binds to neuron - specific nuclear protein and is commonly used to identify neurons . slices from each treatment condition were dual - labeled for arc and zif268 protein to determine colocalization within individual neurons . , all slices were incubated with two washes of 0.1 m pbs for 10 min each before incubation in antibody solution containing anti - mouse alexa 488 and anti - rabbit alexa 594 antibodies ( 1 : 500 dilution each , invitrogen ) for 2 hr in the dark . slices were rinsed in two washes of 0.1 m pbs for 5 min each . after incubation the slices were mounted onto unsubbed slides using ultra cruz mounting medium ( santa cruz ) . finally , the slides were coverslipped and sealed with a thin coat of nail polish around the edges . photomicrographs were taken using a fluorescence microscope ( olympus ) . to determine coexpression of arc and zif268 in the same neurons , separate images were taken of the same field for each protein and then merged using the dp manager ( olympus ) . arc - expressing neurons appear green in color ( alexa 488 ) and zif268-expressing neurons fluoresce red ( alexa 594 ) , thus overlaying the images resulted in coexpressing neurons to appear yellow in color . the exposure time was the same for all the images collected , and any adjustments to the contrast or the brightness of the images were conducted exactly the same for relevant images . protein expression values obtained from western blots were found to be normally distributed , thus the results were analyzed using parametric tests : one - way analysis of variance ( anova ) followed by fisher 's lsd post - hoc comparisons when appropriate . in some cases , pearson correlations were conducted on the normalized optical density values from western blots to investigate the relationship of protein expression in the dorsal hippocampus . significance levels were set at = 0.05 , and data are presented as mean sem . seventy - six male long evans rats were trained in a single 15-min session of pavlovian fear conditioning and later killed at varying time points post training ( 30 min , 60 min , 90 min , 4 hr , 8 hr , 12 hr , and 24 hr ) . all trained groups exhibited equivalent levels of freezing averaged across the 5-min period of cs - ucs presentations ( f(6,69 ) = 1.989 , p > .05 , data not shown ) . protein levels for each experimental condition were expressed as a percentage of the untrained hc animals ' protein expression [ % optical density ( od ) of hc control ] . any animal with a % od score more than 4 standard deviations from the mean was removed from further data analysis ( arc : no outliers ; zif268 : n = 1 from 24 hr group ) . we found that induction of arc protein expression for trained rats was monophasic in the dorsal hippocampus , with a significant increase detected between 30 min and 90 min post training before returning to near basal levels at 4 hr ( figure 2(a ) ) . a one - way anova revealed that the level of arc protein expression was time - dependent following fear conditioning ( f(7,79 ) = 4.265 , p < .001 ) . lsd post hoc comparisons showed that when compared to untrained hc animals , arc protein was significantly increased in trained rats at 30 min ( md = 34.4587 , p < .01 ) , 60 min ( md = 58.0035 , p < .001 ) , and 90 min ( md = 50.8044 , p < .01 ) . in order to determine the localization of arc protein in the dorsal hippocampus , coronal brain slices were collected from rats killed at 90 min after training and prepared for immunohistochemistry . increased arc protein expression was observed in the granule cell layer of the dentate gyrus in the trained animals , whereas only sparse expression of arc protein was detected in this same region in the unstimulated hc control rats ( figures 2(b)2(f ) ) . arc - positive neurons were not evident in other regions of the dorsal hippocampus , such as the ca1 . although we did not quantify the arc - positive neurons in the photomicrographs , the images suggest that the upregulation in arc protein expression is primarily localized in the dentate gyrus . a different temporal pattern was seen in the protein expression profile for zif268 , with a single peak evident at 60 min post training ( f(7,72 ) = 3.228 , p < .01 , see figure 3(a ) ) . lsd post - hoc comparisons revealed that zif268 protein is significantly increased at 60 min when compared to hc animals ( md = 55.2942 , p < .01 ) . in fact , the protein level for zif268 measured at 60 min is significantly higher than all other time points ( 90 min : md = 60.6474 , p < .01 ; 4 hr : md = 71.1209 , p < .01 ; 8 hr : md = 86.4159 , p < .01 ; 12 hr : md = 86.9798 , p < .01 ; 24 hr : md = 69.0759 , p < .01 ) with the exception of 30 min ( md = 32.7638 , p > .05 ) . to establish the localization of zif268 protein in the dorsal hippocampus , coronal brain slices from additional rats killed 30 min post training were collected and incubated with zif268 antibody . in agreement with findings from western blot analysis , conditioned rats showed qualitatively more expression of zif268 protein in the ca1 region of the dorsal hippocampus at 30 min after training , compared to hc controls ( figures 3(b)3(f ) ) . similar increases were evident in the dentate gyrus but not in the ca3 region of the hippocampus ( data not shown ) . some transcription factors ( such as zif268 ) have relatively high basal levels of expression , and this was observed in the unstimulated hc controls ( figures 3(c ) and 3(e ) ) . however , this basal level of zif268 expression was less than the level of zif268 protein induced behaviorally , as measured by western blots ( figure 3(a ) ) and captured visually in photomicrographs ( figures 3(d ) and 3(f ) ) . these images suggest that the increase in zif268 protein measured in western blot analysis is the result of upregulation of zif268 primarily in ca1 and the dentate gyrus ( see figure 4 ) . to validate the temporal changes observed for arc and zif268 protein expression , the same hippocampal samples were assayed for -actin , a constitutively expressed protein , using western blot analysis . the levels of -actin protein in the trained groups did not show significant changes in expression compared to hc controls ( f(7,81 ) = 1.328 , p > .05 ; data not shown ) , indicating that the upregulation in arc and zif268 protein is unique and specific to the behavioral training experience . in summary , the expression profile for these proteins demonstrates an early increase in zif268 expression at 3060 min post training , followed by a gradual monophasic wave in arc induction lasting through 90 min ( figures 2 and 3 ) . the temporal dynamics of these proteins are distinctive and reflect the difference in the functions for arc and zif268 ( i.e. , synapse - specific changes and transcriptional regulation , respectively ) . the delay between the peaks for zif268 and arc proteins corroborates previous research showing that arc is a transcriptional target of zif268 and that multiple genomic responses are activated as a consequence of fear acquisition . using pearson correlation on the western blot optical density values , we investigated the relationship between expression of arc and zif268 proteins in the dorsal hippocampus . correlational analysis indicated a direct moderate relationship between the levels of these proteins in the dorsal hippocampus after fear conditioning ( r(79 ) = 0.244 , p < .05 ) . upon closer inspection , we discovered that this relationship was driven by the positive correlation between arc and zif268 proteins in the animals killed 30 min after training ( r(15 ) = 0.510 , p < .05 ) , as significant correlations between these proteins were not found for the other experimental conditions . other researchers have found arc and zif268 mrna expression in the hippocampus to be correlated following training in hippocampus - dependent tasks , such as the spatial water maze [ 36 , 37 ] . to investigate the colocalization between arc and zif268 proteins in the hippocampus , brain slices collected from trained rats ( sampled 30 and 90 min post training ) and from nave hc rats were dual labeled for these proteins and viewed using epifluorescence microscopy . in the unstimulated hc animals , only a few neurons in the dentate gyrus expressed arc protein ( figure 4(a ) ) ; however , many of these neurons were zif268-positive ( figure 4(b ) ) . the merged image revealed that the arc - positive neurons in the dentate gyrus also expressed zif268 ( figure 4(c ) ) . in the trained rats , more neurons expressed arc protein , with maximal number of arc - positive neurons reached at 90 min ( figures 4(d ) and 4(g ) ) . further , many more neurons in the dentate gyrus expressed zif268 , with qualitatively more zif268-positive neurons shown at 30 min ( figure 4(e ) ) compared to slices collected from nave and 90-min animals ( figures 4(b ) and 4(h ) ) . as was seen in the nave hc animals , arc and zif268 proteins expressed in trained animals are co - localized in the same neurons as depicted in the merged images ( figures 4(f ) and 4(i ) ) . these findings qualitatively replicate the western blot analysis results showing increased arc at 90 min and zif268 around 30 min post training in the dorsal hippocampus . they also extend those findings to suggest that after fear conditioning co - expression of zif268 and arc protein increases . to determine if the time - dependent changes in hippocampal arc and zif268 protein expression were specific to associative learning , rats were assigned to one of four conditions : trained ( t ; n = 14 ) , white noise and context exposure ( wn - cxt ; n = 9 ) , immediate shock ( shk - only ; n = 8) , or unstimulated home cage control ( hc ; trained animals received the same fear conditioning protocol used previously ( figure 1(a ) ) . wn - cxt animals experienced the same training paradigm but with the shock generator turned off , and shk - only rats received shock upon placement in the chamber and then were immediately removed . all rats were killed 60 min post - experience and dorsal hippocampal tissue was processed for western blots . protein levels for each experimental condition were expressed as a percentage of the untrained hc animals ' protein expression [ % optical density ( od ) of hc control ] . arc protein levels did change significantly due to behavioral experience in the training context ( figure 5(a ) ; ( f(3,33 ) = 3.007 , p < both the trained and wn - cxt groups had increased levels of arc protein compared to unstimulated hc controls ( trained : md = 66.3183 , p < .05 ; wn - cxt : md = 71.6449 , p < .05 ) , which was not observed in the shk - only condition ( md = 46.0808 , p > .05 ) . the expression of arc protein in the trained group was similar to arc protein levels observed in the 1-hr group measured in the original time course ( ~60% increase ; figures 2(a ) and 5(a ) ) . a different pattern of protein expression was measured for zif268 , where marked increases were seen only in the trained condition ( figure 5(b ) ) . the overall anova between hc , shk - only , wn - cxt , and trained groups did not meet our statistical criterion ( f(3,33 ) = 1.593 , p > .05 ) . however , the level of zif268 in the trained group was similar to that observed in the 1-hr time group of the original time course ( ~65% ; figures 3(a ) and 5(b ) ) . therefore , we conducted a linear planned comparison between the trained and hc groups which did indicate a significant upregulation of zif268 protein in the trained group ( f(1,33 ) = 4.693 , p < .05 ) . in contrast to both arc and zif268 protein expression , there were no significant changes in protein levels in the loading control -actin across the conditions ( f(3,37 ) = 0.112 , p > .05 ; data not shown ) . the effector ieg arc has been implicated in synaptic plasticity underlying learning and memory . our aim was to extend these earlier findings by investigating the time - dependent expression of arc protein induced by pavlovian fear conditioning and compare it to the expression profile of another ieg , zif268 . we found arc protein to be expressed soon after fear conditioning in the dorsal hippocampus . gradual increases in arc protein were detected by 30 min , and the single peak in the expression profile emerged at 1 - 2 hr post training before returning to baseline levels at 4 hr . arc protein was primarily localized in the granule cell layer of the dentate gyrus . these data indicate that arc protein expression induced in the dorsal hippocampus by fear conditioning is time - dependent and monophasic . arc protein is likely involved in the consolidation of contextual fear memories supported by the hippocampus , since auditory delay fear conditioning is not reliant on the hippocampus [ 26 , 30 ] . the levels of arc protein expression in the dorsal hippocampus were positively correlated with the regulatory transcription factor ieg zif268 . the expression profile for zif268 in the dorsal hippocampus was also monophasic ; however , maximal protein levels were measured at 60 min after fear conditioning , with increased expression seemingly localized to the dentate gyrus and ca1 . in a separate experiment , we investigated if the induction of these proteins was specific to associative learning or was the result of behavioral experience more generally . arc protein expression increased in rats that were trained as well as in those animals exposed to the context and auditory stimuli . delivery of immediate shock did not produce a significant increase in arc , so arc protein expression is not linked to ucs exposure per se . further , we would not predict a significant learning - related increase for the immediate shock condition as this procedure does not result in normal learning . since both training and exposure to the training chamber induced similar levels of arc expression , this effect likely relates to contextual processing . similar alterations in arc mrna in the hippocampus have been observed using catfish analysis for contextual fear conditioned and context - exposed animals . this selective increase in hippocampal zif268 protein only in the trained group and not in shk - only and wn - cxt conditions is similar to the upregulation of zif268 observed during retrieval of a context fear memory relative to cued - fear retrieval or reexposure to a context not paired with shock . the significance of this difference from the pattern seen with arc protein is not yet clear , but perhaps zif268 expression in the hippocampus is more specifically related to the formation of aversive memories . the single phase of arc protein upregulation we observed is similar to forskolin- and ecs- ( electroconvulsive shock ) induced expression of arc mrna [ 1 , 3 , 4 , 40 ] and protein [ 1 , 41 ] , with increased expression measured 30 min to 4 hr post activation and a return to basal levels by 8 or 24 hr . our data are also in accordance with recent ltp in vivo investigations , in which arc antisense oligodeoxynucleotide applied 2 hr , but not 4 hr , post - ltp induction resulted in the reversal of ltp , suggesting that the role of arc protein is time - limited . furthermore , our hippocampal data complement findings from other behavioral paradigms . for example , arc mrna expression in the hippocampus following spatial water maze training peaks at 30 min post training and returns to baseline at 6 hr . we do detect elevated levels of arc protein at 30 min , which may be the product of existing or newly transcribed mrna , a possibility we have not yet investigated or found answered in the literature . however , arc protein at 1 - 2 hr is likely translated from new transcripts synthesized as a consequence of the training experience . this hypothesis is congruent with data suggesting a very rapid turnover of arc , on the scale of minutes to a few hours [ 42 , 43 ] . although spatial water maze and contextual fear conditioning are two different hippocampus - dependent tasks , the mrna and protein time courses for these two studies produce a logical sequence when combined , such that a peak increase for arc mrna at 30 min is followed by maximal arc protein levels at 90 min . multiple waves of increased protein levels may follow a training experience , and the number of phases depends upon the training parameters used [ 44 , 45 ] . for instance , biphasic expression has been measured for other proteins in response to fear conditioning ; however , our data suggest that the expression of arc and zif268 protein in the hippocampus is monophasic following acquisition of conditioned fear . monophasic expression of arc is not necessarily true for all behavioral paradigms , as ramirez - amaya et al . found arc protein to be expressed in two phases in ca1 and ca3 of the hippocampus first phase at 30 min to 2 hr and the second phase at 8 and 24 hr following a single exploration session . in the same study , they found a single wave of arc protein in the dentate gyrus that lasted up to 8 hr post exploration , which is similar to the primarily monophasic expression of arc protein in the dentate gyrus reported here . although the increase was not significant , arc protein did seem to show a moderate increase 24 hr after training . since these animals were killed at varying times during the day , we can conclude that this increase is not due to set circadian patterns in ieg expression . however , memory does seem to have a time - of - day dependence , such that testing at a similar time of day as when training occurred produces better recall . recently , some attention has been given to exploring clock - genes in structures such as the hippocampus that may influence the expression of other proteins and may in effect create time - of - day dependence in memory . circadian fluctuation in protein phosphorylation has been previously observed in the hippocampus for mapk , and this signaling pathway has been implicated in arc translation . whether arc or any ieg is regulated in such a manner is purely speculation at this point , but certainly it is an important consideration for understanding time - dependent changes in protein translation . the post training expression of arc and zif268 proteins in the dorsal hippocampus corresponds to the time window for protein synthesis - dependent memory consolidation . the transient nature of arc expression after training may be related to evidence indicating that arc mrna is targeted for nonsense - mediated mrna decay ( nmd ) as this gene contains two conserved 3-utr introns ( , reviewed in [ 7 , 19 ] ) . nmd is a translation - dependent decay mechanism that likely halts protein expression to produce finite protein levels that are temporally specific to the learning event . along with other degradation pathways , such as ubiquitin - dependent degradation by proteasomes , nmd probably restricts the protein composition to local activated synapses . the notion that the burst of arc protein expression is temporally linked to the learning event is further supported by studies reporting that the level of training - induced arc expression in the hippocampus is coupled with learning performance . for example , guzowski and colleagues demonstrated that the amount of hippocampal arc mrna was positively correlated with an animal 's performance in hippocampus - dependent water maze learning . furthermore , the same study revealed hippocampal arc mrna expression to be correlated with the spatial water maze task ( hippocampus - dependent ) but not with the nonspatial water maze task ( hippocampus - independent ) , indicating that arc expression is associated specifically with learning experiences . presumably , the increased induction of arc and zif268 proteins measured in the present study is the result of contextual processing and associative fear learning , respectively , as the dorsal hippocampus is important for the acquisition and initial consolidation of contextual fear memory . recent investigations on the molecular pathways leading to the induction of arc have focused on brain - derived neurotrophic factor ( bdnf ) , erk , and camp - pka activation . converging evidence indicates that arc is a downstream effector of bdnf activation [ 14 , 43 ] , and pka - dependent arc protein expression can be stimulated either through the activation of nmda receptors or gs - coupled dopamine or -adrenergic receptors [ 41 , 51 ] . recent work by bramham and colleagues suggests that the initiation of arc translation is the result of erk - mnk ( extracellular signal - regulated kinase - mitogen - activated protein kinase - interacting kinase ) signaling in the dentate gyrus , a structure where we observed increases in arc protein . pharmacological blockade of erk with the mek inhibitor u0126 abolishes ltp and arc protein expression ; however , similar results are not observed when mtorc1 ( mammalian target of rapamycin complex 1 ) signaling is inhibited by the application of the protein - synthesis inhibitor rapamycin . comparatively , less is known about the interaction of zif268 and arc following synaptic activation . zif268 is a transcriptional regulator , and it is noteworthy that in our study the peak in zif268 expression at 60 min occurs just prior to the peak in arc at 90 min . the time course for these proteins presented here corroborates earlier evidence that arc is a transcriptional target of zif268 , as the arc promoter has a functional ere ( egr response element ) . additionally , we showed that the levels of hippocampal arc and zif268 were correlated with one another following fear conditioning , which further supports the functional relationship between these two ieg products . work by others similarly found arc and zif268 mrna to be upregulated following spatial exploration , and these iegs are often detected in the same nucleus . these mrnas are correspondingly increased in the hippocampus following spatial water maze training , and their expression profiles are positively correlated within this brain structure . however , the relationship between arc and zif268 is not perfect , as arc protein increases we measured at 30 min are likely not the result of zif268 regulation and are believed to be induced by one of the other pathways implicated in arc expression . further research is needed to determine what role zif268 has in the induction of arc that contributes to synaptic modifications underlying long - term memory . our data suggest that these two proteins interact soon after a learning experience , most likely orchestrating postsynaptic changes to increase synaptic efficacy and support memory formation . in summary , arc and zif268 proteins are transiently increased in the dorsal hippocampus in a manner that suggests that these two proteins work together to support contextual learning in fear conditioning . although we did not establish a causal relationship between associative learning and arc and zif268 protein expression , we have shown that these iegs are consistently upregulated in the hippocampus during the period when memory for context is being consolidated . the time frame of behaviorally - induced arc and zif268 protein in the dorsal hippocampus corresponds to a critical time window in which protein synthesis is required for memory consolidation . further , immunostaining revealed an increase in expression of arc and zif268 protein in the same hippocampal neurons after fear conditioning , suggesting a relationship between arc and zif268 colocalization in consolidation of a contextual fear memory .
pathologic response of tissue to asbestos in vivo gives rise to fibromatoma , granuloma and mesothelioma . we are attempting to develop a model system in vitro using human cells in order to investigate the possible mechanisms responsible for these pathologies . within the first 12 hr of exposure to chrysotile , the fibroblasts showed distinctive morphological changes . cells appeared elongated with occasional vacuolated nuclei and granular cytoplasm . cells showed no other obvious morphological changes by light microscopy and were serially passaged at confluence . the cells with vacuolated nuclei were successfully serially passaged . binucleated cells were first observed 48 hr after passaging . as time in culture increased ( 3 days to 2 weeks ) many cells lost their distinctive bipolar properties and developed " stress striations " and multiple vacuoles in the cytoplasm . multinucleated giant cells ( 2 - 11 nuclei / cell ) with lobate nucleoli became more numerous . with increasing passages , the confluent cell density decreased and cell size increased . cells usually had condensed nucleoli and had lost all control of directional growth . preliminary indications suggest that these in vitro morphological transformations are due -- at least in part -- to a lack of control over cytokinesis.imagesfigure 1 . afigure 1 . bfigure 1 . cfigure 2 . afigure 2 . bfigure 2 . cfigure 3 . afigure 3 . bfigure 3 . cfigure 4 . afigure 4 . b
[ purpose ] the purpose of this study was to examine the reliability of ultrasound measurements by analyzing the relative reliability and absolute reliability when measuring soft tissue thickness around the shoulder joint . [ subjects and methods ] eleven healthy young adults ( 22 shoulders ) participated in this study . thickness of the supraspinatus tendon , subacromial bursa , and biceps tendon was measured on both shoulders . this protocol was performed twice in the same day . the relative reliability of ultrasound measurement was evaluated using the intraclass correlation coefficient for determining the degree of consistency and agreement between two measures . the absolute reliability of the ultrasound measurement was evaluated using the standard error of measurement , minimum detectable change , and bland - altman analysis . [ results ] ultrasound measurements exhibited high relative reliability : intraclass correlation coefficients for the supraspinatus tendon , subacromial bursa , and biceps tendon thickness were demonstrated to be 0.91 , 0.82 , and 0.90 , respectively . bland - altman analyses revealed no significant systematic bias between the repeated measurements for the supraspinatus tendon , subacromial bursa , and biceps tendon thickness . [ conclusion ] these findings suggest that ultrasound measurement for the supraspinatus tendon , subacromial bursa , and biceps tendon thickness exhibited good relative reliability and no systematic errors were detected regarding their absolute reliability .	adhesive capsulitis can be classified into three phases : freezing , frozen , and thawing . patients with the freezing phase are predominantly treated by drug therapy or injection therapy , or both . in many cases , patients experience imperfect treatment effects , which lead to prolonged disease duration of adhesive capsulitis . reported that 40% of adhesive capsulitis patients who had surpassed the average of 44 months from onset experienced residual symptoms3 . hand , et al . reported that 41% of adhesive capsulitis patients who had surpassed the average of 4.4 years from onset experienced either pain or disability in completing daily activities1 . the duration of adhesive capsulitis can be shortened by early initiation of therapy after the cause of disease is identified by a physical therapist . slide disorder in the second shoulder joint , slide disorder of the biceps tendon ( bt ) in the intertubercular sulcus , and rotator interval disorder have been associated with its onset2 , 4 . soft tissues , such as the supraspinatus tendon ( sst ) , subacromial bursa ( sab ) , and bt are thought to be associated with the onset of adhesive capsulitis . for effective treatment of adhesive capsulitis , a physical therapist must objectively evaluate the condition of the soft tissue that caused the disease . the main effect of treatment for the freezing phase of adhesive capsulitis is evaluated by pain , as the condition of the soft tissue that caused this disease can not be objectively evaluated . arthrography , computed tomography , magnetic resonance imaging ( mri ) , and ultrasonography can be used to objectively evaluate the condition of a patient and the effect of treatment . in particular , ultrasonography can be used to simply and immediately scan soft tissue . the spatial and temporal resolution of this technique over a small range is superior to that of mri in measuring soft tissue . when using ultrasonography to assess adhesive capsulitis patients , detecting subacromial bursitis in the early period of the disease is important . similarly , it is important to observe the internal structures of the rotator cuff , bt , and tendon synovial sheath5 . many studies have reported the use of ultrasonography in adhesive capsulitis patients for the assessment of qualitative changes and diagnosis6,7,8 . however , few of them have attempted to use this technique to assess treatment effects in adhesive capsulitis patients9 . sst thickness of the critical zone on ultrasonography was different between the painful side and the non - painful side in patients with adhesive capsulitis10.tendon swelling is interpreted as a focal or more often diffuse increase in tendon thickness8 . during the freezing phase in adhesive capsulitis patients , a small number of studies have evaluated the use of ultrasonography to measure the thickness of soft tissue around the shoulder joint . sst thickness11,12,13,14,15 , supraspinatus muscle belly thickness16 , sab thickness17 , and bt thickness14 , 15 have been investigated . the reliability of ultrasound measurements has been reported by many studies , including those measuring lumbar multifidus thickness18 and the distance of the interval between the tibia and femur19 . however , few studies have examined the reliability of ultrasonography measurements of soft tissue thickness around the shoulder joint12,13,14 . the reliability of these measurements has typically been examined by the intraclass correlation coefficient ( icc ) . icc assesses relative reliability but can not examine absolute reliability that defines the quantity of the measurement errors20 . test and inferential statistics can be nullified by systematic bias that occurs during the planning or study phases . bland - altman analysis is one of the methods used to confirm the presence of systematic bias , and assess the details by examining the quantity and type of error between two measurements21 . the random error of the absolute reliability is examined by standard error of measurement and minimal detectable change . the present study was performed to measure the thickness of the soft tissue around the shoulder joint of young normal subjects by ultrasonography . in addition , the reliability of these measurements was examined by assessing relative and absolute reliability . eleven students were recruited from a technical school and a university physical therapy course . all participants declared their right hand as dominant , and the mean age was 22.8 2.7 years ( means sd ) . exclusion criteria were as follows : shoulder joint pain , humeral proximal edge bone fracture , clavicle bone fracture , shoulder blade bone fracture , traumatic shoulder joint front dislocation , acromioclavicular joint dislocation , rheumatoid arthritis , shoulder arm syndrome , cervical spine lesion , peripheral neuropathy due to diabetes , central nerve obstacle , calcific tendinitis , rotator cuff tear , a bt tear , and shoulder impingement syndrome . ethical approval was obtained from the faculty of health sciences of tokyo metropolitan university ( 14014 ) . sst ( major axis ) and bt ( minor axis ) were scanned twice in both shoulders by ultrasonography . the thickness of sst and sab was measured from the sst ( major axis ) image . a second ultrasound of sst , sab , bt was performed at intervals of about 1 minute after the first scan . ultrasonography was performed using a hitachi eub-7500 ultrasound machine with an 812 mhz linear transducer . the subject s head was in the neutral position , with shoulders and elbow in the neutral position and full extension , respectively . subsequently , the proximal transducer was placed on the acromion , and the distal transducer was placed on the superior facet ( sf ) of the greater tubercle ( fig . 1.image by ultrasonography(a ) image of a supraspinatus tendon and a subacromial bursa by ultrasonography . then proximal transducer was placed the acromion , distal transducer was placed the superior facet ( sf ) of the greater tubercle.(b ) image of a biceps tendon by ultrasonography . then the transducer was moved up and down to the place where subscapularis muscle appears . ) . the subject s head was in the neutral position , with shoulders and elbow in the neutral position and full extension , respectively . subsequently , the transducer was moved up and down to the position at which the subscapularis muscle appears ( fig . each soft tissue thickness measurement of sst , sab , and bt was analyzed using imagej analysis software ( national institutes of health , bethesda , ma , usa ) . measurements of soft tissue thickness around the shoulder joint are often analyzed for the maximum of each thickness13,14,15 . soft tissue thickness was analyzed by the succeeding methods in the present study because previous studies were vague13 , 14 . sst and sab thicknesses were measured as the perpendicular line distance from the first change point of the inclination angle from the top of the greater tubercle ( fig . 2afig . 2.the ultrasound measurement of thickness(a ) the ultrasound measurement of the supraspinatus tendon ( sst ) thickness , the subacromial bursa ( sab ) thickness . sst , sab thickness was measured for perpendicular line distance from the first change point of the inclination angle from the top of the greater tubercle.(b ) the ultrasound measurement of the biceps tendon ( bt ) thickness . bt thickness was measured for perpendicular line distance from the intertubercular sulcus center . ) . bt thickness was measured according to the perpendicular line distance from the intertubercular sulcus center ( fig . image by ultrasonography ( a ) image of a supraspinatus tendon and a subacromial bursa by ultrasonography . then proximal transducer was placed the acromion , distal transducer was placed the superior facet ( sf ) of the greater tubercle . then the transducer was moved up and down to the place where subscapularis muscle appears . the ultrasound measurement of thickness ( a ) the ultrasound measurement of the supraspinatus tendon ( sst ) thickness , the subacromial bursa ( sab ) thickness . sst , sab thickness was measured for perpendicular line distance from the first change point of the inclination angle from the top of the greater tubercle . ( b ) the ultrasound measurement of the biceps tendon ( bt ) thickness . bt thickness was measured for perpendicular line distance from the intertubercular sulcus center . descriptive statistics were presented as the mean ( standard deviation ) for continuous data and as numbers for discrete baseline characteristics . the relative reliability of the ultrasound measurements of sst , sab , and bt thickness was determined by calculating the icc22 . an icc of > 0.80 indicated great to good reliability , 0.700.79 indicated normal reliability , and < 0.60 indicates re - work reliability23 . bland - altman analysis was constructed by plotting the test - retest difference ( ) for two measurements versus the test - retest mean for two measurements . altman analysis indicates the presence or absence of systematic bias in a visual and statistical manner22 , 25 , 26 . the 95% confidence interval ( ci ) of mean difference ( ) of two measurements , which was used to determine the presence of systematic bias , was calculated as follows22 , 25,26,27:95% ci of d= t ( sd / n)where sd is the standard deviation for all the observations from test sessions 1 and 2 , n is the sample size , and t is the value obtained from the t - table with degrees of freedom . if 0 is included within the 95% ci , no significant systematic bias between the measurements can be inferred24,25,26 . when a systematic error was not recognized , standard error of measurement ( sem ) and minimal detectable change ( mdc ) were calculated . sem was calculated as follows26,27,28:sem = sd ( 1-icc ) the mdc is calculated as mdc , which is the ci of 95% of the mdc . mdc95 was calculated as follows19 , 27,28,29,30:mdc95=sem 1.96 2 the mdc indicates the limit area that the amount of change in repeated two measurements results from measurement error29 . if the change of measurement is within mdc , the measurement error between repeated measurements can be inferred . if the change of measurement is greater than mdc , the true change between repeated measurements can be inferred30 . detailed characteristics of the participants and the soft tissue thickness around the shoulder joint are shown in table 1table 1.characteristics of the participantscharacteristicparticipantsgendermale7female4age ( years)22.8 2.7supraspinatus muscle tendon thicknessright ( mm)4.73 0.8left ( mm)4.47 0.6subacromial burse thicknessright ( mm)0.66 0.3left ( mm)0.66 0.2biceps tendon thicknessright ( mm)2.21 0.6left ( mm)2.31 0.6 . sst thickness values were as follows : right , 4.73 0.8 mm and left , 4.47 0.6 mm . sab thickness values were as follows : right , 0.66 0.3 mm and left , 0.66 0.2 mm . bt thickness values were as follows : right , 2.21 0.6 mm and left , 2.31 0.6 mm . the results of the relative reliability of sst , sab , and bt thickness are shown in table 2table 2.relative and absolute reliability of a supraspinatus tendon , an subacromial bursa , a biceps tendon by ultrasound measurement ( n=22)soft tissueicc ( 95%ci)semmdc95bland - altman analysisdsdd95%ci of dsst0.91 ( 0.800.96)0.090.260.070.310.070.21sab0.82 ( 0.630.92)0.070.180.030.160.040.10bt0.90 ( 0.790.96)0.080.230.010.260.100.13icc : intraclass correlation coefficient ; 95%ci : 95% confidence interval ; sem : standard error of measurement ; mdc : minimal detectable change ; mdc95 : 95%ci of mdc ; sst : supraspinatus tendon ; sab : subacromial bursa ; bt : biceps tendon . the relative reliability of sst , sab and bt thickness was rated as excellent ( icc=0.91 ; 95% ci=0.800.96 ) , good ( icc=0.82 ; 95% ci=0.630.92 ) , and excellent ( icc=0.90 ; 95% ci=0.790.96 ) , respectively . icc : intraclass correlation coefficient ; 95%ci : 95% confidence interval ; sem : standard error of measurement ; mdc : minimal detectable change ; mdc95 : 95%ci of mdc ; sst : supraspinatus tendon ; sab : subacromial bursa ; bt : biceps tendon the results of the absolute reliability of sst , sab and bt thickness are shown in table 2 . the 95% cis of sst , sab , and bt thickness were 0.070.21 , 0.040.10 , and 0.100.13 , respectively . sems of sst , sab , and bt thickness were 0.09 , 0.07 , and 0.08 , respectively . mdcs95 of sst , sab , and bt thickness were 0.26 , 0.18 , and 0.23 , respectively . in the present study , standard methods were used to evaluate the relative and absolute reliability of ultrasound measurements of sst , sab , and bt thickness . ultrasound measurements in the present study exhibited good relative and absolute reliability , demonstrating a high degree of agreement between the 1st and 2nd measurements , small measurement error , and lack of systematic bias . establishing the reliability of a tool for the adequate measurement of sst , sab , and bt thickness is an essential prerequisite before the tool is adopted as a standard measurement in patients with acute adhesive capsulitis . the iccs of sst , sab , and bt thickness were 0.91 , 0.82 and 0.90 , respectively . when relative reliability was evaluated using the criteria outlined by kuwahara et al . , sst , sab , and bt thickness were rated as good to great23 . therefore , the relative reliabilities of ultrasound measurement for sst , sab , and bt thickness were demonstrated to be high . supraspinatus ( tendon ) thickness data from a healthy adult ( icc=0.91 ; 95% ci=0.800.97)16 and a university student from hong kong ( icc=0.92 ; 95% ci=0.900.98)12 indicated excellent reliability . in the present study , a detailed landmark was not set when scanning sst , sab , and bt via ultrasonography . by contrast , in previous studies , the supraspinatus ( tendons ) was scanned after a landmark was set13 , 17 . sst and infraspinatus tendons are in close proximity to the greater tubercle of the humerus . therefore , when sst was scanned alone by ultrasonography , it was necessary to scan on the sf of the greater tubercle . if a detailed landmark was set before scanning sst via ultrasonography , it was very difficult to scan the sf of the greater tubercle . the shoulder joint consists of plural joints , including the glenohumeral , acromioclavicular , sternoclavicular , and scapulothoracic joints . it is difficult to attain the same alignment of the shoulder joint in all subjects . therefore , the sst on the sf of the greater tubercle was scanned via the monitor of the ultrasonography device . the result of relative reliability in the present study demonstrated good relative reliability ; therefore , it is thought that the ultrasound measurement by the method of this study was effective . bland - altman statistics for the ultrasound measurement of sst , sab , and bt thickness indicated no significant systematic bias between the repeated measurements . the mean differences between the two testing sessions were all close to 0 and the 95% ci included 0 . it is difficult to resolve a systematic bias by repeated measurements under the same conditions . test and inferential statistics are nullified by a systematic error that occurs during the study planning and experimental phases31 . the present study had no significant systematic bias . sem and mdc95 assess measurement errors between repeated measurements and determine whether changes occur between these repetitions . in the present study , the mdcs95 of sst , sab , and bt thickness were 0.26 , 0.18 , and 0.23 , respectively . if the changes in repeated measurements for sst , sab , and bt thickness were > 0.26 , 0.18 , and 0.23 mm , respectively , the change was real within the 95% ci . a treatment effect may be indicated if the changes in sst , sab , and bt thickness , as detected by ultrasound measurement , were more than the mdc95 values for acute adhesive capsulitis patients . the results of reliability testing in the present study showed that ultrasonography may be a useful measurement tool for assessing the condition of acute adhesive capsulitis patients and the effects of treatment . ultrasonography has many advantages , including the ability to scan soft tissue simply and immediately . therefore , we suggest that physical therapists evaluate acute adhesive capsulitis patients via ultrasound measurements to determine the severity of their condition and assess treatment effects . as a result , first , the validities of the measurement for sst , sab , and bt thickness were not examined . second , the small number of participants adversely affected the power of the study . finally , this study only evaluated healthy young adults . the final objective of the present study is to form a standard ultrasound measurement in patients with acute adhesive capsulitis . as the majority of patients with adhesive capsulitis are elderly , the reliability of ultrasound measurements should be examined in elderly individuals in the future . the present study examined the reliability of ultrasound measurement for the sst , sab , and bt thickness . the results demonstrated that the relative reliability of these measurements ranges from good to great , and a systematic error was not detected in absolute reliability testing . these examinations of reliability suggest that it is possible to use ultrasound measurements as an evaluation for the condition of acute adhesive capsulitis patients and to assess treatment effects .
acute tumor lysis syndrome ( tls ) is a condition resulting from rapid destruction of tumor cells and subsequent massive release of cellular breakdown products . it has been described following the treatment of many hematologic and solid malignancies . however , spontaneous tls has rarely been described . here we report a case of spontaneous tls that occurred in a patient with a treated maxillary squamous cell carcinoma ( scc ) presenting with diffuse liver metastases , which is an infrequent site of distant metastases .	acute tumor lysis syndrome ( tls ) is a constellation of metabolic disturbances that can develop in rapidly proliferating tumors , mainly hematologic malignancies , or more rarely in solid tumors after initiation of cytotoxic therapy . it is characterized by the release of large amounts of potassium , phosphate , and nucleic acids into the systemic circulation . this can lead to hyperuricemia , hyperphosphatemia , hyperkalemia and hypocalcemia , which can cause lactic acidosis , acute renal failure , cardiac arrhythmias , seizures , muscle cramps , tetany , syncope , and possibly sudden death . tls has been reported to occur in very few cases of solid tumors without any prior therapy . here we report an unusual case of a maxillary squamous cell carcinoma ( scc ) who presented a few months after treatment of the primary disease with diffuse liver metastases and tls . the patient is a 53-year - old man who started to complain of progressive left cheek pain , nasal obstruction and epistaxis . ct scan of the head and neck showed a left maxillary sinus mass invading the medial and anterior walls of the sinus , extending into the left nasal cavity and soft tissues of the cheek and eroding the floor of the orbit . biopsy from the tumor revealed infiltrating squamous cell carcinoma arising from an inverted papilloma with focal high - grade dysplasia . the patient underwent a radical maxillectomy that showed infiltrating squamous cell carcinoma of 2.8 2 2 cm originating from an inverted papilloma with presence of vascular and perineural invasion and negative margins of resection . after surgery , the patient received adjuvant chemoradiation of 66 gy to the tumor bed and 50 gy to the upper neck area . at the end of treatment , abdominal ultrasound was requested and revealed multiple hypoechoic liver nodules that are suspicious for metastases ( fig . ct - guided core biopsy of one of these lesions was performed and showed high - grade carcinoma with focal positivity for ck8/18 and no staining for high - molecular - weight cytokeratin , compatible with a metastatic poorly differentiated carcinoma similar to the previous pathology . four days later , the patient presented to the emergency room with a decrease in the level of consciousness and abdominal pain . laboratory investigations revealed a bun of 144 mg / dl ; creatinine , 6.4 mg / dl ; uric acid , 20.9 mg / dl ; potassium , 7.6 mg / dl ; phosphorus , 11.8 mg / dl ; calcium , 6.2 mg / dl ; alp , 734 iu / l ; ggt , 621 iu / l ; and lactate dehydrogenase ( ldh ) , 1,000 u / l ( table 1 ) . the clinical picture and the rapidly progressive disease , the acute deterioration in electrolytes , and kidney function are all in favor of an acute tls . he was also given one dose of rasburicase 8 mg , but he deteriorated rapidly and passed away the following day from tls . tls is characterized by hyperphosphatemia , hyperuricemia , hyperkalemia , hypocalcemia , lactic acidosis , and acute renal failure . hyperuricemia is the result of purine degradation and may lead to precipitation of uric acid crystals in the collecting tubules in the kidney , resulting in obstructive nephropathy . hyperkalemia is due to potassium release from the cytoplasm and may lead to cardiac arrhythmias and cardiac arrest . hyperphosphatemia , caused by nucleoprotein degradation , may cause precipitation of calcium phosphate in the renal tubules . hypocalcemia follows the precipitation of calcium phosphate in the tissues and may cause neurologic and muscular symptoms . patients at highest risk for acute tls are those who have a large tumor burden or rapidly proliferating tumors , mainly hematologic malignancies , such as leukemia and lymphoma . acute tls is a metabolic complication of chemotherapy : cytotoxic therapy can induce cytolysis of neoplastic cells and release of intracellular substances , which can precipitate this syndrome . it is more rarely seen in solid tumors , and it has been reported to occur after therapy . a review of the literature by kalemkerian et al . found 25 cases of patients with solid tumors who developed acute tls after treatment . these tumors are usually bulky with multiple metastatic sites , and acute tls was described to develop after recent treatment . only a few cases of spontaneous tls in solid tumors have been described without any prior therapy ( table 2 ) . in this case , acute tls may be due to cell turnover rather than to a treatment effect . crittenden and ackerman were the first to report a case of disseminated adenocarcinoma of the gastrointestinal tract with renal failure and high levels of uric acid . we reported the first case of tls secondary to liver metastases from a primary maxillary scc . on the other hand , the incidence of lymphadenopathy at diagnosis is very low because the maxillary sinuses have a limited lymphatic supply . reviewed 220 patients who were treated for nasal and paranasal sinus carcinoma between 1975 and 1994 with a minimum follow - up of 4 years and found that only 9 patients ( 4.3% ) developed distant metastasis . in a recent study published by tanvetyanon et al . , the medical records of patients with inverted papilloma ( ip ) and scc treated during 1999 - 2007 were retrospectively reviewed . four biopsy specimens were described as scc arising in ip , one case was read as carcinoma in situ arising in ip and one as ip with areas of severe dysplasia . liver metastases were described in one case and were discovered at the time of local recurrence along with lung metastases 33 months after diagnosis . in conclusion , our case is unique in so far as the patient developed liver metastases , which is a rare location for distant metastases from maxillary tumors , even without developing local or locoregional recurrence , and in the development of spontaneous tls , which is an unusual finding in solid tumors and was never reported in sinus scc . spontaneous tls should be anticipated in solid tumors with a large volume of metastatic disease .
curricula for residents on rotations through intensive care units are necessarily abbreviated . the selection ( and omission ) of topics can be informed by assessment of perceived needs . a curriculum can not , however , be formed exclusively from the top - scoring needs . topics that are encountered exclusively in the critical care unit ( such as brain death ) must be included .	the mind is never passive ; it is a perpetual activity , delicate , receptive , responsive to stimulus . you can not postpone its life until you have sharpened it . whatever interest attaches to your subject - matter must be evoked here and now ; whatever powers you are strengthening in the pupil , must be exercised here and now ; whatever possibilities of mental life your teaching should impart , must be exhibited here and now . that is the golden rule of education , and a very difficult rule to follow . ( alfred north whitehead , presidential address to the mathematical association , january 1916 ) peets and colleagues report on a strategy for selecting content for inclusion in a critical care curriculum for residents . the authors constructed a three - domain classification of common clinical problems and asked resident trainees and attendings to score each problem according to the threat to life , to frequency and to reversibility . the scales were organized to give greatest weight to greater life - threat , higher frequency and ease of reversibility . the authors report strong concurrence between the product of domain scores of resident trainees and of their supervising attending physicians . in their conclusion , the authors assert that their process is widely applicable and ' can facilitate creation of a reliable and valid curriculum ' . it is unsurprising that residents and their teaching staff should have similar assessments of the three objective features listed . for example , brain death which appears at the bottom of the priority list is irreversible by definition . if any resident or attending scored brain death as anything other than not reversible , it would be at once surprising and problematic . similarly , the frequency of the condition of brain death in the intensive care unit ( icu ) studied and the degree to which brain death threatens life are not matters for debate . what is of greater concern , however , is that the methodology advanced by the authors results in brain death being placed at the very bottom of the needs assessment . the authors state in their key messages that their tool ' will provide content validity for any curriculum ' . herein lies the greatest problem with the methodology : it overvalues those curricular elements that focus on reversible conditions . the unfortunate fact is that many patients are admitted to the icu with conditions that either will not respond to critical care or are terminal , or both . curricula that fail to forthrightly confront this reality perpetuate costly and quixotic efforts to cure where care would be more appropriately directed towards comfort and dignity . the same scoring logic relegates end - of - life decision - making , arguably one of the most important activities in any icu , to a level of importance below that of obstetrical complications . the critical question left unaddressed in the methodology proposed for selecting curricular elements is whether the topics most highly ranked can be uniquely and best learned in the icu . for example , topics such as shock , seizure and drug overdose are highly ranked , but are also frequently encountered in the emergency department . in contrast , the management of acute and fulminant hepatic failure , and the strategies that reverse those conditions or else indicate the need for transplantation , can only be learned in the icu . yet acute and fulminant hepatic failure ranks just above brain death in the needs assessment listing . in summary , the authors should be complimented for conducting a needs assessment and also for reporting strong concurrence between trainees and supervisors . the report should serve as a basis for ensuring that acute and critical care topics are covered within a comprehensive curriculum spanning the entire training period . the fact that a condition might not be reversible should not diminish its importance in the icu curriculum . on the contrary , one might reasonably argue that brain death , fulminant organ failure and end - of - life decision - making ought to be pushed near the top of the priority list . if these topics are not explored during the icu rotation of the trainees , then where ?
cutaneous metastases from internal malignancies are uncommon and occur in 0.6%10.4% of all patients with cancer . in most cases , cutaneous metastases develop after the initial diagnosis of the primary internal malignancy and late in the course of the disease . skin tumors are infrequent in asian population and cutaneous metastases are quite rare . cutaneous metastases carry a poor prognosis with average survival of few months . in the present five - year study 1924 malignant tumors were screened which included only nine cases of cutaneous metastatic deposits . a wide range of site and clinical presentations including nodules , plaques , and ulcers was noted . histopathological findings were significant and corresponded with the primary internal malignancy . cutaneous metastases from breast carcinoma ( 44.4% ) were the most common finding followed by non - hodgkin lymphoma and renal cell carcinoma ( 22.2% each ) and carcinoma cervix ( 11.1% ) . the aim of our study is to classify the cutaneous metastases and to evaluate their clinicopathologic and immunohistochemical correlation with the primary tumor .	cutaneous metastasis can be defined as the spread of a tumor from the site of its primary origin to the skin . skin metastasis may be the first sign of an advanced cancer or an indicator of cancer recurrence [ 2 , 3 ] . up to 9% of patients with cancer may develop skin metastases , while metastasis may develop more than 10 years after initial cancer diagnosis . a wide morphologic spectrum of clinical appearances has been described in cutaneous metastases including nodules , plaques , papules , tumors , and ulcers . while carcinomas are the most common type of cancer to metastasize , sarcomas , lymphomas , and leukemias also represent a substantial percentage of all skin metastases . the relative frequencies of metastatic skin disease in each sex correlate with the frequency of different types of primary cancer . thus women with the skin metastases have the following distribution in decreasing order of primary malignancies : breast , ovary , oral cavity , lung , and large intestine . in men , the distribution is as follows : lung , large intestine , oral cavity , kidney , breast , esophagus , pancreas , stomach , and liver . generally , cutaneous metastases herald a poor prognosis with average survival time of a few months . in the present five - year study , patients diagnosed with an internal malignancy including hematolymphoid neoplasms , registered between march 2009 and march 2014 in the pathology department , were consecutively screened . the h&e stained histopathological sections of skin biopsies received in the pathology department were reevaluated . the inclusion criteria were cases of cutaneous metastatic deposits with or without known primary malignant tumor . physical and dermatologic examination details were obtained from the patient files and histopathology requisition forms . the clinical presentation , site , and histopathological details , especially those suggesting the primary tumor site , were evaluated along with the secondary morphological changes in the skin tissue . immunohistochemistry was performed on all except for one case of cutaneous metastases and correlation with the primary internal malignancy was done . in the present five - year study , a total of 1924 malignant tumors were screened which included nine cases of cutaneous metastatic deposits . the cutaneous metastases were seen more in females ( 5 out of 9 patients ) . the four male patients had skin metastases from renal cell carcinoma and from non - hodgkin lymphoma ( 2 cases each ) . the age range was found to be 3072 with mean age 60 years . a wide range of clinical presentations and regional localizations was noted . plaque and nodule were the most frequent clinical presentation ( 4 cases each out of 9 ) followed by ulcer ( 1 case out of 9 ) . the size of the skin lesions varied from 0.25 cm to 5.0 cm . the regional localization in cases of breast carcinoma included chest ( 2 cases ) , chest and abdomen ( 1 case ) , and face , scalp , and trunk ( 1 case ) . a case of non - hodgkin lymphoma showed widespread skin deposits on face , scalp , and trunk while another case showed localized deposits on abdomen only . a single case of carcinoma cervix showed skin deposits on thigh ( table 1 ) . the duration of time after which the cutaneous metastases developed was variable and ranged from 10 months to five years . in majority of the cases , patients had prior history of a primary internal malignancy . the morphological patterns and microscopic appearances suggested the likely tissue of origin . in cases of cutaneous metastases from carcinoma breast , the histologic examination revealed invasion of dermis and subcutis by groups , cords , and nests of tumor cells . er / pr positivity was seen in 3 out of 4 cases of cutaneous metastases from carcinoma breast . the metastatic deposits which were negative for er / pr had previous reports of er / pr negativity in the primary tumor too . the deposits of renal cell carcinoma showed presence of tumor cells in glandular configuration or in nests . ihc ( cd-10 ) was applied and confirmed the diagnosis in both cases ( figures 2 and 3 ) . the deposits of non - hodgkin lymphoma showed diffuse presence of atypical lymphoid cells in the dermis and subcutis . the atypical lymphoid cells showed finely stippled chromatin , inconspicuous nucleoli , and sparse cytoplasm . there was no evidence of epidermotropism which ruled out the possibility of primary cutaneous lymphoma . leucocyte common antigen ( cd 45 ) was positive in both of the cases ( figures 4 and 5 ) . the deposits of carcinoma cervix showed presence of tumor cells arranged in groups and nests . the tumor cells were large and showed vesicular nuclei and moderate amount of eosinophilic cytoplasm . as the epidermis was not involved , the differentiation of cutaneous metastatic deposits from primary squamous cell carcinoma was possible . moreover , a prior history of squamous cell carcinoma of cervix three years back also suggested metastatic skin deposits . in all the cases of cutaneous metastatic deposits , the main challenge for the pathologist is to exclude the possibility of primary skin neoplasms ( including benign and malignant adnexal tumors ) and inflammatory conditions of skin especially in cases of skin deposits of nhl . cutaneous metastases occur infrequently and are rarely present at the time the cancer is initially diagnosed . cutaneous metastases occur in 0.6%10.4% of all patients with cancer and represent 2% of all skin tumors . in the present five - year study , only 9 cases out of a total of 1924 patients with internal malignancies presented with cutaneous metastases , thus showing a prevalence rate of approximately 0.5% which is near to the lower limit of the reported range . cutaneous metastases may either be the initial manifestation of an internal malignancy or represent recurrent neoplastic disease . in the present study , all the nine patients had prior history of primary internal malignancy , thus representing the recurrence of the primary tumor . a landmark study by brownstein and helwig from 1972 found the most common tumors to metastasizing to the skin were breast , lung , colorectal , and melanoma . while gul et al . in their study found that most common cancer types metastasizing to skin were breast , colon , and ovary in females and lung and colon cancers in males . in the present study , the order of cutaneous metastases from internal malignancy was carcinoma breast ( 4/9 cases ) and carcinoma cervix ( 1/9 case ) in females while it was renal cell carcinoma and non - hodgkin lymphoma ( 2/9 cases each ) in males . cutaneous metastases from carcinoma breast were the most common finding which is in concordance with the above mentioned studies . according to basu and mukherjee , gynecological malignancies rarely give rise to metastatic deposits on the skin . skin metastasis from uterine cervical carcinoma is a rare event with the reported incidence ranging from 0.1 to 2% . our study also included a single case of cutaneous metastasis from carcinoma cervix which is quite rare . skin metastases of renal cell carcinoma are not easily identified because of the low suspicion index for these skin lesions , which usually mimic common dermatologic disorders . skin metastases of renal cell carcinoma have been reported to occur in around 3% of renal tumors and are more common in males . in our study we reported two cases of cutaneous metastases from renal cell carcinoma and both the patients were males . cutaneous metastases were most frequently ( 2.6% ) seen in cases with hematological malignancies in a study done by gul et al . , while in the present study cutaneous metastases from nhl and rcc were the most common tumors in males . it has been observed that many carcinomas spread through the lymphatic route to areas having common lymphatic drainage as that of the primary site . in the present study as well , the skin deposits from breast carcinoma were mainly localized to the chest wall . scalp is relatively a rare site for the localization of skin metastases . in our study , two out of nine cases showed cutaneous metastases on scalp . according to lookingbill et al . , the most common presentation of cutaneous metastatic deposits is multiple nodular lesions . contrary to that nodules and plaque were the most common clinical presentation in our study . metastatic carcinomas are usually differentiated from primary skin carcinomas because of the latter 's typical histological patterns , the epidermal connection , intraepidermal / intra - adnexal ( in situ component ) tumor , or the presence of a benign counterpart [ 3 , 6 ] . in cases where distinction between metastatic and primary skin tumor is difficult , a variety of immunohistochemical staining panels can be helpful [ 1317 ] . in our study too , ihc applied in eight out of nine cases correlated well with the primary tumor . tumor markers are becoming increasingly important in breast cancer research because of their impact on prognosis , treatment , and survival . the er and pr markers used in skin deposits from breast carcinoma confirmed the primary tumor . according bauer et al . , er , pr , and her-2 neu negative breast cancers affect younger women and were more aggressive and these women had poorer survival regardless of stage . in two cases , histopathological diagnosis was clear cell carcinoma which on immunostaining with cd 10 was confirmed as metastatic renal cell carcinoma . similarly , lca positivity confirmed the metastatic deposits of nhl in other two cases . to conclude , cutaneous metastases occur infrequently and that internal malignancy rarely presents with skin involvement . however , early diagnosis is necessary which may have profound effect on patient management and survival .
background . medical nutritional therapy is the most important method for normalizing glucose levels in pregnancy . in this setting , there is a new problem to consider relating to migrants , their personal food preferences , and ethnic , cultural , and religious aspects of their diet . we compared maternal and fetal outcomes between two multiethnic groups of pregnant women , one adopting a food plan that included dishes typical of the foreign women 's original countries ( the ethnic meal plan group ) , while the other group adopted a standard meal plan . findings . to develop the meal plan , each dish chosen by the women was broken down into its principal ingredients . the quantity of each food was given in tablespoons , teaspoons , slices , and cups , and there were photographs of the complete dish . the group treated with the ethnic meal plan achieved a better metabolic control at the end of the pregnancy and a lower weight gain ( though the difference was not statistically significant ) . as for fetal outcome , the group on the ethnic meal plan had babies with a lower birth weight and there were no cases of macrosomia or lga babies . conclusions . this preliminary study indicates the positive effect of an ethnic approach to diet on the outcome of pregnancy .	gestational diabetes ( gdm ) , that is , carbohydrate intolerance first recognized and/or diagnosed during pregnancy , is the most common metabolic complication of gestation , with a prevalence estimated at around 14% [ 2 , 3 ] . obesity , family history of diabetes , and belonging to certain ethnic groups increase the risk of gdm [ 411 ] . if the disorder is not properly monitored and treated , it can cause severe complications in both mother ( including preeclampsia , cesarean delivery , glucose intolerance , or type 2 diabetes after delivery ) and child ( macrosomia , hypoglycemia , hyperbilirubinemia , adolescent obesity , glucose intolerance , and diabetes ) [ 2 , 4 , 12 , 13 ] . immigration rates have recently increased in italy , and immigrants now account for about 4% of the resident population ; 48% of the immigrant population consists of women , and 65% of them are of reproductive age . some studies have demonstrated a tendency for adverse outcomes of pregnancy among immigrant women from countries with high rates of diabetes [ 58 ] . immigrants differ in their cultural background and eating habits , so we aimed to assess such women 's compliance with dietary restrictions and the possible benefit , in terms of maternal and fetal outcome , of adopting a nonstandard , ethnic - based approach to their diet . for this pilot study , twenty pregnant immigrant women with gdm followed up at the metabolic disease and diabetology unit of padova university between january and june 2008 were enrolled . the study protocol complied with the helsinki declaration and was approved by the local ethics committee , and written informed consent was obtained from all participants . screening for gdm was done with a glucose challenge test ( gct ) between weeks 24 and 28 of gestation , and the diagnosis was confirmed with a 100 g oral glucose tolerance test ( ogtt ) as recommended by the 4th international workshop conference on gdm . the women enrolled were randomly assigned to two groups : one adopted a standard meal plan ( smp ) prepared according to the ada guidelines , the other an ethnic meal plan ( emp ) ( table 1 ) . all the women were monitored to achieve a good metabolic control , that is , fasting plasma glucose ( fpg ) < 5.3 mmol / l and 1 h postprandial plasma glucose ( 1 h pppg ) < 7.2 mmol / l , and nurses taught them to monitor their own blood glucose levels . the pregnant women on diet treatment performed 2 measurements per day , measuring fasting and 1 h postprandial glucose on alternate meals over the course of a week . the women on insulin therapy performed self - glucose monitoring four times a day ( fasting and 1 h after breakfast , lunch , and dinner ) . insulin treatment was started when fpg and/or 1-hour pppg exceeded the above level in more than one measurement . all gdm women were followed up for metabolic and obstetric purposes until delivery . for maternal characteristics and outcome , we considered age , prepregnancy body mass index ( bmi , kg / m ) , time of gdm diagnosis , hba1c ( when gdm was diagnosed and at delivery ) , percentage of patients on insulin , weight gain , timing and mode of delivery , and hypertensive disorders . for fetal outcome , we considered birth weight , infants large or small for gestational age ( lga , sga ) , and fetal composite outcome ( hypoglycemia , neonatal asphyxia , respiratory distress syndrome , and hyperbilirubinemia , hypocalcemia ) and fetal malformations . babies were lga if their birth weight was above the 90th percentile and sga if it was below the 10th percentile according to population - specific standard growth tables . macrosomia was diagnosed for a fetus weighing more than 4000 g. a dietary assessment was conducted to determine whether a woman 's intake of essential nutrients was adequate and whether she was eating excessively and to identify foods she avoided , as well as food intolerances or allergies . a meal plan food models , using measures in cups , glasses , and bowls , proved helpful props when teaching appropriate serving sizes . the two groups received different meal plans : group 1 adopted the smp for gdm according to the ada guidelines ; group 2 adopted the emp , which included typical foods of the women 's home countries , identified using a photographic atlas ( dietmeter and photographic atlas , scotti bassani ) [ 1719 ] . dishes were broken down into the various ingredients , shown raw and cooked . due to difficulties in using kitchen scales , measures such as cups , or the two meal plans had the same nutrient composition ( smp : cho 53% , l 28% , p 18% fiber 26 g ; emp : cho 55% , l 28% , p 17% , fiber 21 g ) , and energy intake was from 1800 to 2200 kcal , depending on prepregnancy bmi . adherence to the diet was measured using a 24-hour food intake recall method and scored as 0 for an intake more than 20% higher than prescribed , 1 if the intake was 1020% higher ; 2 if it was consistent with the plan or up to 10% lower . the intake was calculated in individual tables , based on the inran nutritional tables , 2000 version . hba1c was measured using standard hplc ; the normal range assumed for healthy pregnant women was 4.05.5% ( 2037 mmol / mol ) . data are given as means standard deviations and were compared using student 's t - test for unpaired data or for paired data when comparisons were drawn at different times in the same sample . the groups were compared for categorical data or frequency of an event using the test with yates ' correction . the ethnic distribution in emp group was chinese 1 ; filipino 1 ; moroccan 1 ; nigerian 3 ; romanian 4 ; bangladeshi 1 . in smp group , was chinese 1 ; moroccan 1 ; nigerian 1 ; romanian 4 ; sudanese 1 , bangladeshi 1 , hungarian 1 , with no difference between the two meal plan groups . table 2 shows the women 's clinical and metabolic characteristics , and maternal and fetal outcomes . mean age , prepregnancy bmi , and time of gdm diagnosis were comparable for the two groups . the emp group had better fpg , 1hpppg , and hba1c values than the smp group . weight gain was lower , though not significantly , in the emp group ( figure 2 ) . the newborns ' birth weight was slightly higher in the smp group , which also included more lga ( 3 versus 0 , p < 0.001 ) and macrosomic babies ( 2 versus 0 ) than the emp group . no fetal complications or congenital malformations were seen in either group ( table 2 ) . adherence to the meal plans was better in the emp group , with 7 women scoring 1 or 2 ( good adherence ) , as opposed to 2 women in the smp group . italian data on pregnancy indicate a relatively poor outcome of such pregnancies due to difficulties in accessing care and following medical recommendations for cultural and social reasons . some immigrant populations are also at higher risk of type 2 diabetes and gestational diabetes [ 2 , 8 , 10 ] . better standards of living in host countries often have negative effects on the health of immigrant populations , partly because foreign citizens acquire eating habits and lifestyles unsuited to their genetic profile . regarding gdm , the problem is exacerbated by a genetic predisposition of african , afro - american , and hispanic races to develop diabetes [ 4 , 8 , 9 ] . nutritional therapy for immigrant women should take the social , cultural , and religious value attached to food by the various ethnic groups into account . in our study , the main problems the women had with their prescribed diet were related to difficulties in changing their eating habits ; problems with managing a meal plan and weighing the foods ; doubts concerning which foods to choose ; difficulties in achieving the right nutritional balance . on the other hand , many studies have confirmed the important influence of dietary treatment on the outcome of gdm . the real difference between the two meal plans we used lies in how the diet and examples of typical dishes are presented . the meal plan containing new elements adapted to different ethnic and cultural needs , with photos and practical domestic units of measure to illustrate the quantities of each food , plus lists of alternative foods and photos showing the foods raw and cooked proved useful and effective for our immigrant women with gdm , since adherence was better among the patients adopting the emp . better adherence coincided with better glycemic control , a more normal weight gain , and a better pregnancy outcome , that is , the birth weights were lower , and there were no lga or sga babies in the emp group . the weakness of our study lies in the small numbers of patients considered but , to the best of our knowledge , this is the first study on the feasibility and efficacy of customized dietary treatment for immigrant gdm patients . this pilot study indicates the positive effect of an ethnic approach to diet on the outcome of pregnancy . the new methods introduced in our study could be considered a valid approach to the nutritional management of immigrant pregnant women with gestational diabetes mellitus . it points out that , when prescribing a diet for immigrant women , their different traditional eating habits should be borne in mind . a study with an adequate number of women chosen in accordance with the power calculation is necessary and useful to confirm these preliminary data .
adequate cognitive functioning is essential for daily activities . when there is an insult to the brain , cognitive abilities can suffer , which , in turn , produce substantial medical and functional impairment . advances in neurobiology , circuit neuroscience , and clinical assessment technology are converging in a manner that holds promise for the development of new pharmacological agents for cognitive enhancement in neuropsychiatric disease .
backgrounda bulk of contraceptives on the market is women - oriented today . the aim of this study was to investigate the effect of a medicinal herb , acacia nilotica on various parameters of male fertility using a rat model.methodsmale wistar rats ( n = 40 ) were randomly divided in to two groups . one group received acacia nilotica , while the other acted as controls . ten animals from each group were sacrificed after 16 weeks . treatment was withdrawn for the remaining animals for 8 weeks . blood was collected for hormonal analysis . the testis was removed for histological examination , while epididymal spermatozoa were retrieved for motility and morphological analysis . the data were analyzed using anova and bonferroni post hoc test . a value of p<0.05 was considered statistically significant.resultssperm motility , progressive motility and sperm concentration significantly decreased in treated animals compared to the controls ( p<0.05 ) . withdrawing the treatment did not restore these parameters ( p<0.05 ) . abnormal sperm morphology significantly increased in both the treated and treatment withdrawn groups when compared to the controls ( p<0.05 ) . testosterone concentrations were significantly lower in the treated group when compared to the controls ( p<0.05 ) and no significant differences were observed between the controls and the treated animals when treatment was withdrawn . histological observations showed that acacia nilotica treatment disrupted semeniferous tubule architechture and consequently the spermatogenesis process.conclusionthese results show that acacia nilotica severely affects sperm morphology , progressive motility and sperm concentration irreversibly in wistar rats .	therefore , numerous methods are being used to reduce the total fertility rate in both men and women , especially in developing countries . most of the poorest countries , especially in sub - saharan africa , are characterized by rapid population growth ( 1 ) . it is estimated that half of all conceptions are unplanned and half of the resulting pregnancies are undesired ( 2 ) . in most cases , half of the unintended pregnancies are due to failure to use contraception , and the other half to difficulties with contraceptive usage or method failure ( 3 ) . in poor nations , contraceptive use is further limited by restricted access to many available methods , both economically and culturally . undesired pregnancies result in unwanted children who disproportionately suffer from poverty and neglect ( 4 , 5 ) . women have a wide range of contraceptive choices ranging from daily oral medications to intrauterine devices implanted every 5 years to sterilization . research and family planning organizations have , for a long time , focused upon female methods of contraception because women bear a disproportionate portion of the health and economic consequences of childbearing and rearing . the consequence of this long neglect of developing acceptable and reliable male contraceptives in poor developing countries has been the lack of or lower participation of males in family planning . recently , it has become apparent that neglecting men in matters of family planning is a losing strategy with adverse consequences for both men and women ( 6 ) . men around the world and in africa are showing willingness to actively participate in family planning despite the drawbacks of the currently available male contraceptive methods . . the disadvantages of these methods are that vasectomy is not readily reversible and condoms have a high typical failure rate . there are many references to plants with antifertility properties in the literature ( 7 , 8) and numerous plants and their products have also been shown to possess antispermatogenic activities ( 9 , 10 ) . the aim of this study was to investigate the effect of a medicinal herb , acacia nilotica ( a. nilotica ) , on various male fertility parameters . since the toxicity of this plant is not yet known , a rat model was used . dried pods of a. nilotica were obtained from a local forest in blantyre , malawi in the same winter season between may and july . botanical identification was done at the malawi national herbarium and botanical gardens and the specimen was given voucher number 1mal . the dried pods of the plant were grounded into homogenous thin powder using an electric grinder . the crude extract was prepared by making a suspension of 100 g of the powder in 500 ml of water to make an aqueous extract . the suspension was left to stand overnight at room temperature , with constant agitation and then filtered . the solution was concentrated in a vacuum at 40c using a rotor vapour until crystals were formed . sexually mature male wistar rats ( 284430 g body weight ) were housed in standard rat cages and maintained under standard conditions ( 12 hr light / dark cycle ; 253c temperature ; 35% 60% relative humidity ) , provided with a standard laboratory chow and water ad libitum . one group received a. nilotica treatment ( 200 mg / kg rat , daily ) , while the other group acted as a control . a. nilotica treatment was withdrawn for the remaining animals for 8 weeks before being sacrificed . after the last day of treatment , the body weight of each animal was recorded and they were killed along with control animals . testis and cauda epididymis of each animal were excised from the surrounding tissue and blotted free of blood for weighing . the cauda epididymis was separated and minced using a pair of small scissors , to release the sperm into 10 ml of warmed physiological saline . the sperm suspension was placed in an incubator at 37c for 10 min prior to total motility and progressive motility assessment . the aliquot of the sperm suspension was further diluted 5 times with warm physiological saline and then placed on makler counting chamber and motile sperm were counted under a light microscope . progressive and total sperm motility were expressed as a percent of motile sperm of the total sperm count . for sperm count , five counts per sample were made and averaged . the smears were left to air dry before stained by rapid diff staining ( australian biostain , australia ) . briefly , smears were submerged for 6 one - second dips in rapid diff fixative . the smears were then dipped six times for one second each in rapid diff stain 1 followed by six dips in rapid diff stain 2 . finally , the slides were rinsed in phosphate buffer ( ph = 6.8 ) and air dried . the right testes from both control and experimental groups were dissected out and fixed in formal saline . the tissues were processed for histological examination and paraffin sections were stained with hematoxylin and eosin and qualitative microscopic examination was made . serum concentration of total testosterone were determined using the coat - a - count total testosterone assay kit ( diagnostic products co. , usa ) , per manufacturer 's instruction . assay sensitivity was 0.2 ng / ml . the results were analyzed on the prism 4 statistical program ( graphpad , usa ) . one - way anova ( with bonferroni post hoc test if p<0.05 ) was used for statistical analysis . dried pods of a. nilotica were obtained from a local forest in blantyre , malawi in the same winter season between may and july . botanical identification was done at the malawi national herbarium and botanical gardens and the specimen was given voucher number 1mal . the dried pods of the plant were grounded into homogenous thin powder using an electric grinder . the crude extract was prepared by making a suspension of 100 g of the powder in 500 ml of water to make an aqueous extract . the suspension was left to stand overnight at room temperature , with constant agitation and then filtered . the solution was concentrated in a vacuum at 40c using a rotor vapour until crystals were formed . sexually mature male wistar rats ( 284430 g body weight ) were housed in standard rat cages and maintained under standard conditions ( 12 hr light / dark cycle ; 253c temperature ; 35% 60% relative humidity ) , provided with a standard laboratory chow and water ad libitum . one group received a. nilotica treatment ( 200 mg / kg rat , daily ) , while the other group acted as a control . a. nilotica treatment was withdrawn for the remaining animals for 8 weeks before being sacrificed . after the last day of treatment , the body weight of each animal was recorded and they were killed along with control animals . testis and cauda epididymis of each animal were excised from the surrounding tissue and blotted free of blood for weighing . the cauda epididymis was separated and minced using a pair of small scissors , to release the sperm into 10 ml of warmed physiological saline . the sperm suspension was placed in an incubator at 37c for 10 min prior to total motility and progressive motility assessment . the aliquot of the sperm suspension was further diluted 5 times with warm physiological saline and then placed on makler counting chamber and motile sperm were counted under a light microscope . progressive and total sperm motility were expressed as a percent of motile sperm of the total sperm count . for sperm count the smears were left to air dry before stained by rapid diff staining ( australian biostain , australia ) . briefly , smears were submerged for 6 one - second dips in rapid diff fixative . the smears were then dipped six times for one second each in rapid diff stain 1 followed by six dips in rapid diff stain 2 . finally , the slides were rinsed in phosphate buffer ( ph = 6.8 ) and air dried . the right testes from both control and experimental groups were dissected out and fixed in formal saline . the tissues were processed for histological examination and paraffin sections were stained with hematoxylin and eosin and qualitative microscopic examination was made . serum concentration of total testosterone were determined using the coat - a - count total testosterone assay kit ( diagnostic products co. , usa ) , per manufacturer 's instruction . the results were analyzed on the prism 4 statistical program ( graphpad , usa ) . one - way anova ( with bonferroni post hoc test if p<0.05 ) was used for statistical analysis . the sperm motility results showed that rats treated with a. nilotica had significantly low total motility compared to the control rats ( p<0.05 ) . withdrawing the treatment did not improve the total motility back to control level ( p<0.05 , table 1 ) . progressive motility significantly decreased in the a. nilotica treated group as well as the treatment withdrawn group when compared to the controls ( p<0.05 , respectively , table 1 ) . sperm concentration significantly decreased in a. nilotica treated group , as well as when treatment was withdrawn , compared to the control group ( p<0.05 , respectively , table 1 ) . abnormal sperm morphology significantly increased in both the treated and the treatment withdrawn groups when compared to the controls ( p<0.05 , respectively , table 1 ) . on the other hand , testosterone levels were only significantly lower in the treated group when compared to the controls ( p<0.05 , table 1 ) . the effects of acacia nilotica treatment on total sperm parameters and plasma testosterone levels in wistar rats values in rows are meanssem . means followed by the same letter in the row do not differ significantly . if the letter in the same row differs from that of the control , then p<0.05 in the testicular histology study , seminiferous tubules of control animals showed clear organization of cells at various stages of spermatogenesis with clear spermatozoa maturation occurring near the lumen ( figure 1 ) . in treated rats , transverse sections ( ts ) of the seminiferous tubules of control rats transverse sections ( ts ) of the seminiferous tubules of rats treated with acacia nilotica this study showed that treating rats with a. nilotica for 16 weeks leads to decreased total sperm motility , as well as progressive motility . withdrawing the treatment for 8 weeks the study also shows that a. nilotica treatment in rats reduces testosterone level ; however , when the treatment is withdrawn , testosterone levels return to normal values . this was indicated by histological study of the testis of extract - fed animals , which exhibited partial depletion of the spermatogenic process . in which carica papaya seed extract caused androgen deprivation in male rats leading to alterations in the internal milieu of the cauda epididymis ( 7 ) . development of effective , safe and acceptable male contraceptive is challenging . to be embraced by males , it must have no effect on libido or sexual function , and it must be reversible . the approach to the development of a male contraceptive can be either to inhibit the production of sperm , interfere with sperm function and structure , interrupt sperm transport , interrupt sperm deposition or prevent sperm - egg interaction ( 9 , 10 ) . finding an oral herbal contraceptive would allow couples control their fertility without consulting a health worker , which in turn would markedly increase the number of couples practicing family planning . other advantages of such a contraceptive would include the familiarity rural people in africa have with herbal medicines , the fewer side effects associated with herbal preparations , their availability from local sources , and protection of privacy ( 11 ) . trees of a. nilotica in africa are already recognized for their use in traditional medicine as well as a gum with potential food and pharmaceutical applications . it has been reported that the bark of a. nilotica is able to treat headaches , while the leaves can treat constipation ( 12 ) . from this study , it is evident that a. nilotica severely affects male animal fertility parameters .
purposethese days , mesenchymal stem cells ( mscs ) have received worldwide attention because of their potentiality in tissue engineering for implant dentistry . the purpose of this study was to evaluate various growth inducing factors in media for improvement of acquisition of bone marrow mesenchymal stem cells ( bmmscs ) and colony forming unit - fibroblast ( cfu - f).materials and methodsthe mouse bmmscs were freshly obtained from female c3h mouse femur and tibia . the cells seeded at the density of 106/dish in media supplemented with different density of fetal bovine serum ( fbs ) , 1 , 25-dihydroxyvitamin ( vd3 ) and recombinant human epidermal growth factor ( rhegf ) . after 14 days , cfu - f assay was conducted to analyze the cell attachment and proliferation , and moreover for vd3 , the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2h - tetrazolium ( mts ) assay was additionally conducted.resultsthe cell proliferation was increased with the increase of fbs concentration ( p<.05 ) . the cell proliferation was highest at the density of 20 ng / ml rhegf compared with 0 ng / ml and 200 ng / ml rhegf ( p<.05 ) . for vd3 , although the colony number was increased with the increase of its concentration , the difference was not statistically significant ( p>.05).conclusionfbs played the main role in cell attachment and growth , and the growth factor like rhegf played the additional effect . however , vd3 did not have much efficacy compare with the other two factors . improvement of the conditions could be adopted to acquire more functional mscs to apply into bony defect around implants easily .	recently , stem cell is the much - anticipated research area especially in cell - based therapeutic strategies,1 and we can obtain the stem cell from several tissues like bone marrow , umbilical cord , adipose and periodontal ligament.2,3 as already known , bone marrow derived mesenchymal stem cells ( bmmscs ) have self - renewal capacity and pluripotency so that they can differentiate into other types of cells such as bone cells , cartilage cells and adipose cells.1,4,5 however , the main issue of bmmscs is how we can get more cells effectively . the isolation and proliferation of mscs from mouse , human and other species has been more difficult owing to the cultures contaminated by the unexpected non - mscs for several passages.6,7 a number of techniques have been developed to isolate mscs and improve the purity.6 based on the technique , a novel way was introduced that change the media composition.8 numerous factors can contribute to the cell attachment and growth . fetal bovine serum ( fbs ) which is a ubiquitously used essential component for animal cell culture contains hormones , growth factors and adhesion - promoting molecules like fibronectin and vitronectin.9,10 another interesting factor , 1 , 25-dihydroxyvitamin ( vd3 ) , regulates cell proliferation and differentiation,11 and its effects may vary with different types of cells.12 moreover , the growth factor , like recombinant human epidermal grow factor ( rhegf ) is also thought as the enhancer of the growth rate.13 however , till now , no research has completely analyzed the most suitable density of the above three factors for mbmmsc culture . the present study was therefore conducted to focus on the mbmmscs ' response to different concentration of fbs , vd3 and rhegf in order to make the conclusion regarding which density is the best condition for the cell growth . six to eight weeks specific pathogen free ( spf ) grade c3h female mouse weighting 20 - 25 g were used in this study and fed on a standard diet . the animal husbandry and all procedures performed by the institutional animal care and use committee of the seoul national university school of dentistry and conducted according to national institute of health guideline . all study procedures were approved by the institutional review board at seoul national university school of dentistry ( irb no . then , the animal skeleton was rinsed with 70% ethanol , followed by making an incision close to the hind limbs where they attach to the trunk and striping the skin by pulling toward the foot , which is cut at the anklebone . this eliminates further contact of the hind limb with the animal 's fur , which is a source of contamination . then , the hind limbs from the trunk of the body were dissected by cutting along the spinal cord with care not to damage the femur . dissected limbs were stored on ice pack in phosphate - buffered saline ( pbs , gibco by life technologies , grand island , ny , usa ) containing dish and further dissection of the hind limb was performed under the hood . the muscle and connective tissue were removed from the tibia and the femur with forceps.14 harvesting of the bone marrow was done in a hood using appropriate sterile technique as follows : firstly , wipe the bone with napkin which is moist by 70% ethanol . then , cut the ends of the tibia and femur just below the end of the marrow cavity using scissors . insert a 27-gauge needle attached to a 10-ml syringe containing serum free media ( sfm)-(without fbs ) into the cut end of the bone . the reason for using sfm is to except the interference of fbs for we test the effects of different density of fbs . flush the marrow plug out of the cut end of the bone with sfm and collect into a 15-ml tube on ice . filter the cell suspension through a 70 m nylon filter mesh ( bd falcon , bd biosciences - discovery labware , frankin lakes , nj , usa ) to remove any bone trivial debris or muscle and cell clumps.14 the cells were treated with ack lysing buffer ( lonza , wakersville , md , usa ) for 5 min and centrifuged at 1500 rpm ( 4 ) for 5 min so that the interference of other cells especially hematopoietic cell can be avoided . the yield and viability of cells were determined by trypan blue ( gibco by life technologies , grand island , ny , usa ) and the cell number was counted using hemocytometer . the mbmmscs were primary seeded at a density of 10 in 60 mm dish and cultured in different conditional media : ( 1 ) alpha minimum essential medium ( -mem)(gibco by life technologies , grand island , ny , usa ) supplemented with different density ( 0% , 1% , 5% , 10% , 20% ) of fetal bovine serum ( fbs)(equitech - bio , inc . , kerrville , texas , usa ) , 1% antibiotic - antimycotic ( gibco by life technologies , grand island , ny , usa ) , 1% l - glutamax ( gibco by life technologies , grand island , ny , usa ) , 0.1% 2-mercaptoethanol ( gibco by life technologies , grand island , ny , usa ) ; ( 2 ) alpha minimum essential medium ( -mem ) supplemented with 10% fbs media combined with different density ( 0 nm , 1 nm , 10 nm , 100 nm ) of 1 , 25-dihydroxyvitamin ( vd3)(cayman chemical , ann arbor , mi , usa ) , 1% antibiotic - antimycotic , 1% l - glutamax , 0.1% 2-mercaptoethanol ; ( 3 ) alpha minimum essential medium ( -mem ) supplemented with 10% fbs media combined with different density ( 0 ng / ml , 20 ng / ml , 200 ng / ml ) of recombinant human epidermal growth factor ( rhegf ) ( prospec , east brunswick , nj , usa ) , 1% antibiotic - antimycotic , 1% l - glutamax , 0.1% 2-mercaptoethanol . the cells were incubated at 37 in 95% humidified air and 5% co2 . after 72 hours , non - adherent cells were removed by washing with phosphate - buffered saline ( pbs ) twice and fresh media was added . after 14 days , the cultures were washed with pbs twice and stained with 1% toluidine blue solution in 2% paraformaldehyde ( pfa , wako pure chemical industries , ltd . , osaka , japan ) , then incubated at room temperature ( rt ) on rocker for overnight . a cell cluster that had more than 50 cells was counted as a colony under microscopy . all experiments were repeated in triplicate . the proliferation rate of mbmmscs cultured in the presence of 1 , 25-dihydroxyvitamin ( vd3 ) was measured by 3-(4,5-dimehylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2h - tetrazolium ( mts ) assay . briefly , freshly isolated mbmmscs ( 10 cells each ) were seeded in 96-well plastic culture plates and incubated in 200 l -mem supplemented with different density ( 0 nm , 1 nm , 10 nm , 100 nm ) of vd3 for 1 day , 4 days , 7 days , 10 days , 14 days . cell proliferation was determined using celltiter 96 aqueous one solution reagent ( promega , madison , wi , usa ) . at every point , after removal of the media , wash with pbs ( twice ) and add 100 l sfm with 20 l mts solution and incubated for 1 - 4 hours at 37. formazan absorbance was read at 490 nm using a plate reader and software accent / mts . all data were expressed as mean sd . differences between groups were analyzed by using the one - way anova ( spss version 21.0 for windows 7 ) with a post hoc turkey test and were considered statistically significant at p - values of less than .05 . mbmmscs were harvested from c3h mouse and after 72-hour culture , primary cells attached and non - attached cells were eliminated by pbs washing with media changing . after the mbmmscs cultured in different density of fbs media , vd3 media and egf media for 14 days , they represented different growth rate respectively . 1 ) showed that cells cultured in 0% , and 1% fbs gradually died and did not form the colony . with the fbs concentration increasing , the proliferation rate also improved simultaneously and in 5% fbs , the colony began to form . in 10% and 20% fbs , the cell proliferation was significantly increased and the 20% dishes had the higher proliferation than 10% dishes . 3 ) showed that the proliferation of cells cultured in different density of vd3 . the cell number increased with increasing of vd3 concentration from 0 nm to 10 nm and the cell number did not change any more in 100 nm , but the difference was not statistically significant . 5 ) showed that the media containing with egf accelerates the cell growth , and the media supplemented with 20 ng / ml egf had the highest cell proliferation compared with 0 ng / ml and 200 ng / ml egf . 6 ) also indicated that the 20 ng / ml egf dish had the most colony numbers compared with the other two dishes . fig . 7 showed that the cell proliferation of 20% fbs was significantly higher than that of 10% fbs and 5% fbs ( p<.05 ) . 8 showed that the difference between the growth in 0 nm , 1 nm , 10 nm and 100 nm vd3 was not statistically significant ( p>.05 ) . 9 showed that compared with remaining two groups , the cells cultured in 20 ng / ml egf exhibited significant increase in cell viability ( p<.05 ) . the additional proliferation assay was conducted to further analyze the function of vd3 on mbmmsc . mts result ( fig . 10 ) showed that the cell increased over time . at every point , the vd3 did not play the significant effect ( p>.05 ) . at day 10 , in this study , we described cellular response to the culture media containing different density of fbs , vd3 and rhegf . first , since the mouse bone marrow mesenchymal stem cell is still difficult to many researchers,7 in this study , we introduced a novel way of supplementing different components into the culture media so that the primary isolated cell number and cell growth rate can be increased . previous studies only paid attention to modify the isolation protocol such as employing a centrifugation step,16 the method of plastic adherence,17 immunodepletion way,18 frequent media changing , and changing the plating density.19 additionally , we choose the fbs , vd3 , rhegf which are very common and can be easily obtained . conventionally , the mscs were cultured in fbs containing media which was considered as the basal growth medium in animal cell culture.20 tropel et al.20 indicated that when the mouse bone marrow stem cells were cultured without serum , they showed senescence signs and died in few days . li et al.21 published the article about rat bone marrow mesenchymal stem cells cultured in different density of fbs . in the research , they used 10% , 11% and 15% fbs culture medium and finally they found that a serum concentration of 11% is preferential for bmsc propagation . we designed 0% , 1% , 5% , 10% , 20% five different density of fbs culture medium . in our experiment , the cells cultured in 0% and 1% fbs media were almost impossible to grow , and along with adding more fbs , the cells were gradually grown more quickly and amplified . in 10% fbs media , the cell proliferation was visibly increased and 20% fbs media had stronger effects on cell growth than 10% fbs media . these results indicate that the mbmmscs could not grow well without fbs and fbs play a pivotal role in cell culture . 1 , 25-dihydroxyvitamin d3 ( vd3 ) , the hormonally active form of vitamin d , is a member of a lipophilic family of ligands and is essential for human metabolism.22 except for its role in calcium and skeletal homeostasis , the evidence increased about its potential for osteoblast differentiation23 and anti - proliferation.24,25 in previous study , artaza et al.26 cultured mouse c3h 10t1/2 multipotent mesenchymal cells ( mmcs ) in 10 nm , 25 nm , 50 nm , 100 nm and 500 nm or without vd3 culture medium for 4 days and determined cell proliferation . their results indicated that starting at 25 nm , vd3 induced a statistically significant reduction in cell number reaching a plateau at 100 nm . okuno et al.12 also did the research about culturing a murine myogenic cell line c2c12 in 0 nm , 1 nm , 10 nm , 100 nm vd3 containing medium and the results showed that vd3 inhibited the proliferation of c2c12 myoblasts in a dose - dependent manner over 72 hours . so in our study , we used the vd3 to investigate its effect on mouse bone marrow stem cells . we also used 0 nm , 1 nm , 10 nm , 100 nm four different vd3 containing medium . in our study , the cell number slightly increased with increasing of vd3 density , but it no longer increased after exceeding the density of 10 nm . for a more in - depth analysis , except for colony forming unit assay but the data did not reveal statistical significance which means that vd3 had little effects on mbmmscs . in previous research , several growth factors have been introduced in stem cell culture like vegf , fgf-2 , pdgf and egf.20,27,28,29 in our study , we choose the egf for it is inexpensive and easy to manipulate.28 very recently , you et al.30 did the research about whether epidermal growth factor gene - transfected mesenchymal stem cells ( egf - msc ) would accelerate fibroblast migration and proliferation . the study suggested that egf increased expression of cell adhesion molecules and had a positive influence on cell migration and proliferation . in another study , ml-1 fgf2 to culture mouse epidermal neural crest stem cells ( epi - ncscs ) for 7 days . the results suggest that the combination of egf - fgf2 stimulates the proliferation and improves the neuronal potential of epi - ncscs . in order to explore if egf alone can has the same stimulating effect to cell growth , we planned this study and the results of our study were in consistent with previous studies . in our study , we used 0 ng / ml , 20 ng / ml , 200 ng / ml egf , then the egf represented the promoting role indeed and revealed 20 ng / ml was the best condition for cells . all these experiments possessed to aim at getting more cells at the initial stage . after treated by three different media , the cells showed different reactions . collectively , the results of this study indicate that fbs play the main role in primary cell acquisition and proliferation . growth factors like rhegf just have the additional effects and vd3 has little effect on cell proliferation . based on this study , people can do extensive research about changing other media components to increase primary isolated cell number and cell proliferation .
impairment of the protein c pathway plays a central role in the pathogenesis of sepsis . administration of recombinant human activated protein c ( rhapc ) may correct the dysregulated anticoagulant mechanism and prevent propagation of thrombin generation and formation of microvascular thrombosis . furthermore , it may simultaneously modulate the inflammatory response . it is likely that the beneficial effect of rhapc observed in experimental and clinical studies of severe sepsis results from a combination of mechanisms that modulate the entangled processes of coagulation and inflammation . this review presents an analysis of the various mechanisms of action of rhapc in sepsis .	in all patients with sepsis there is abundant activation of inflammatory pathways , which results in demonstrable circulating levels of inflammatory cytokines and chemokines , activated inflammatory cells and other markers of increased inflammatory activity . these abnormalities range from subtle activation of coagulation , which can only be detected by sensitive markers of coagulation factor activation ; to more marked activation , which may be detectable based on a small decrease in platelet count and subclinical prolongation of global clotting times ; and finally to fulminant disseminated intravascular coagulation ( dic ) , which is characterized by simultaneous widespread microvascular thrombosis and profuse bleeding from various sites . septic patients with severe forms of dic may present with thromboembolic disease or clinically less apparent microvascular failure that predominantly presents as multiple organ dysfunction . interestingly , there is a tight , bidirectional relationship between activation of inflammation and coagulation , in which inflammatory activity results in activation of coagulation but activated coagulation proteases can also affect inflammatory pathways . activated protein c ( apc ) appears to play a central role in the pathogenesis of sepsis and associated organ dysfunction . there is ample evidence that insufficient functioning of the protein c pathway contributes to the derangement of coagulation observed in sepsis . the circulating zymogen protein c is activated by the endothelial cell bound thrombomodulin once this is activated by thrombin . apc acts in concert with its co - factor , protein s , and can proteolytically degrade the cofactors va and viiia , which are essential for coagulation ; apc is therefore an effective anticoagulant . the endothelial protein c receptor ( epcr ) not only accelerates activation of protein c several fold but it also serves as a receptor for apc , and binding of apc to epcr may amplify its anticoagulant and anti - inflammatory effects . a recent study demonstrated that exposure of cultured endothelial cells to apc results in release of microparticles that contain epcr , but the relevance of that observation to coagulation or inflammation is not yet clear . the protein c system . the solid arrows indicate the mechanisms by which the protein c system is impaired in sepsis . in patients with sepsis the apc system malfunctions at virtually all levels . first , plasma levels of the zymogen protein c are low or very low because of impaired synthesis , consumption and degradation by proteolytic enzymes such as neutrophil elastase [ 10 - 12 ] . furthermore , significant downregulation of thrombomodulin caused by pro - inflammatory cytokines such as tumour necrosis factor- and interleukin-1 has been demonstrated , resulting in diminished protein c activation . low levels of free protein s may further compromise the functioning of the protein c system . in plasma , 60% of the co - factor protein s is complexed to the complement regulatory protein c4b - binding protein ( c4bbp ) . increased plasma levels of c4bbp , which occur as a consequence of the acute phase reaction in inflammatory disease , may result in relative protein s deficiency , which further contributes to a procoagulant state during sepsis . although it has been shown that the -chain of c4bbp ( which mainly governs binding to protein s ) is not much affected by the acute phase response , support for this hypothesis comes from studies conducted in baboons in which infusion of c4bbp in combination with a sublethal dose of escherichia coli resulted in a lethal response , with severe organ damage due to dic . finally ( but importantly ) , in sepsis epcr has been shown to be downregulated , which may further adversely affect the function of the protein c system . apart from these effects , sepsis may induce resistance to apc by other mechanisms that are partly dependent on a sharp increase in factor viii levels ( released from endothelial cells ) and partly due to as yet unidentified mechanisms . administration of apc in a baboon model of intravenous e. coli administration resulted in survival of all animals , whereas all control animals in the same experiment died . a similar beneficial effect was observed in rabbits with meningococcal endotoxin shock . in a rat model of septic shock , administration of apc prevented tumour necrosis factor- mediated hypotension , probably caused by modulation of the nitric oxide response . in patients with severe sepsis , administration of recombinant human activated protein c ( rhapc ) resulted in remarkable improvement in microcirculatory perfusion . conversely , experiments conducted in baboons in which the protein c pathway was blocked with monoclonal antibodies resulted in complete lethality in an otherwise sublethal model of bacteraemia . in this same model , blockade of the epcr also resulted in a more severe response to sublethal e. coli bacteraemia . in addition to these observations in experimental sepsis models , apc was shown to have antithrombotic properties in experimental thrombosis models in dogs , rabbits and baboons . interestingly , apc may also affect fibrinolysis by inhibiting plasminogen activator inhibitor type 1 ( a fibrinolytic inhibitor ) . in a rat model of dic , apc was shown to block activity of plasminogen activator inhibitor type 1 , and other experiments demonstrated the ability of apc to enhance clot lysis in vivo . more definitive proof of the beneficial effect of rhapc in severe sepsis comes from clinical studies . these studies are reviewed in detail in other reviews included in this supplement [ 27 - 29 ] and are not discussed in detail here . however , it may be of interest to review the evidence that rhapc acts as an anticoagulant agent in these studies . first , it should be noted that the selected dose of rhapc was based on the effect on d - dimer levels in a phase ii clinical trial . indeed , in the pivotal prowess ( recombinant human activated protein c worldwide evaluation in severe sepsis ) trial , septic patients who were treated with apc exhibited a significant decrease in d - dimer levels as compared with placebo control individuals . d - dimer levels dropped by 25% after 2 days of apc administration , which was in contrast to a 10% increase in placebo - treated patients . in a more detailed analysis of coagulation activation upon administration of rhapc , it was clearly demonstrated that markers of thrombin generation sharply dropped almost immediately after initiation of the apc infusion . second , subgroup analyses of trials including patients with severe sepsis demonstrated that patients with the most extreme coagulation abnormalities benefit the most from treatment with rhapc . the relative risk reduction in mortality among patients with sepsis and dic who received apc was 38% , as compared with a relative risk reduction of 18% observed among patients with sepsis who did not have dic . interestingly , the dynamics of coagulation abnormalities in the first days after intensive care unit admission for severe sepsis , including the response of the protein c system , is a strong predictor of outcome . it is difficult to assess whether this anticoagulant effect of rhapc translates into an antithrombotic effect . in the recently concluded xpress ( xigris [ drotrecogin alfa ] and prophylactic heparin in severe sepsis ) study , all patients with severe sepsis received rhapc but were also randomly assigned to receive prophylactic heparin or placebo . the main conclusion of this study was that heparin was not equivalent to placebo and might have a beneficial effect on 28-day mortality . of note , this advantageous effect of heparin was completely due to a greater incidence of death and thrombotic adverse events in the patients receiving heparin but who were randomly assigned to placebo ( in other words , those who stopped heparin during the trial ) . there was a markedly low incidence of venous thromboembolism in this study compared with previous reports ( the incidence did not differ between placebo patients and patients receiving heparin ) , even though all patients underwent screening ultrasound for venous thrombosis at around day 6 of admission . in addition , a report of small series of patients with severe sepsis who were treated with rhapc demonstrated a lack of thrombotic obstruction of haemofiltration circuits , even in the absence of heparin or other anticoagulants . apart from the systemic response , there may be a differential localized effect of rhapc on coagulation . the localized effect of apc appears to be particularly marked in the pulmonary compartment . in experiments involving unilateral instillation of endotoxin into healthy individuals , systemic administration of rhapc resulted in a marked reduction in bronchoalveolar activation of coagulation . this observation may be relevant because the vast majority of patients with severe sepsis in the various trials had a pulmonary source of infection . an interesting novel finding is the modulatory influence of alveolar epithelial cells on the protein c pathway . it is likely that the underlying mechanism involves shedding of epcr and thrombomodulin by metalloproteinases . evidence that apc acts as an important mediator in the systemic inflammatory response in sepsis comes from experiments showing that blocking the protein c pathway in septic baboons exacerbated the inflammatory response . in contrast , administration of apc ameliorated the inflammatory activation that occurred upon intravenous infusion of e. coli . similar experiments in rodents yielded identical results and demonstrated a beneficial effect on inflammatory effects in various tissues . support for the notion that apc has anti - inflammatory properties comes from in vitro findings , demonstrating an apc - binding site on monocytes that may mediate downstream inflammatory processes . it also received support from experiments showing that apc can block nuclear factor-b nuclear translocation , which is a prerequisite for increased levels of pro - inflammatory cytokines and adhesion molecules . these in vitro findings are supported by in vivo studies in mice with targeted disruption of the protein c gene . in these mice with genetic deficiencies of protein c , endotoxaemia was associated with more marked increases in pro - inflammatory cytokines and other inflammatory responses than in wild - type mice . it is likely that the effects of apc on inflammation are mediated by epcr , which may mediate downstream inflammatory processes . binding of apc to epcr influences gene expression profiles of cells by inhibiting endotoxin - induced calcium fluxes in the cell and by blocking nuclear factor-b nuclear translocation . the epcr - apc complex itself can translocate from the plasma membrane into the cell nucleus , which may be another mechanism of modulation of gene expression , although the relative contributions of this nuclear translocation and cell surface signalling are unclear . some studies have also suggested that epcr binding of apc can result in activation of protease activated receptor ( par)-1 and thereby affect cytokine responses . in contrast , other experiments demonstrated that a significant physiological role for activation of par-1 by apc to be less probable . soluble epcr ( the extracellular domain of the cell - associated epcr shed from the cell surface by the action of an inducible metalloproteinase ) can bind to proteinase 3 , which is an elastase - like enzyme . the resulting complex binds to the adhesion integrin macrophage 1 antigen ( mac-1 ) . of considerable interest is that the crystal structure of epcr is remarkably similar to the structure of the mhc class 1/cd1 family of proteins , the majority of which are involved in inflammation . blocking the epcr with a specific monoclonal antibody aggravated both the coagulation and the inflammatory response to e. coli infusion . apart from the influence of apc on cytokine levels , remarkable effects of the agent on leucocyte chemotaxis and adhesion of circulating leucocytes to the activated endothelium have been demonstrated . this was confirmed in a hamster endotoxaemia model at concentrations of rhapc that preclude a significant anticoagulant effect . the localized effect of apc in the lung has also been shown to exhibit these anti - inflammatory properties . apc was shown to inhibit the expression of platelet - derived growth factor in the lung , which may reflect a potential mechanism underlying this localized effect . also , apc was shown to protect against disruption of the endothelial cell barrier in sepsis , probably by interfering with epcr and par-1 on endothelial cells [ 56 - 58 ] . finally , apc can inhibit endothelial cell apoptosis , which also appears to be mediated by binding of apc to epcr and to require par-1 . signalling through this pathway can affect bcl-2 homologue protein , which can inhibit apoptosis , and further suppresses p53 , which is a pro - apoptotic transcription factor . inadequate functioning of the protein c system , and in particular apc , plays a central role in the pathogenesis of sepsis . attempting to restore the function of this pathway in patients with sepsis appears to be a rational approach and is supported by the beneficial effect of apc in experimental models of sepsis and in clinical studies . apart from its evident effect on the coagulation system and the ability of rhapc to correct the deranged coagulation system in severe sepsis , a series of pleiotropic modulating effects of apc on inflammatory cytokines and cells as well as protective effects on disrupted endothelium have been reported . it should be noted that many of the effects on inflammatory cells and pathways , as well as the cytoprotective effect , have mostly been demonstrated in vitro and sometimes at inordinately high concentrations of apc . the relevance of these findings to the human in vivo situation and their importance to the treatment of sepsis remain to be established . although the relative importance of the anticoagulant effect versus the inflammation - modulating effect of rhapc is not clear , it is tempting to hypothesize that a combined effect is responsible for the benefit from rhapc . indeed , strategies aimed at restoring physiological pathways with less marked effects on inflammation ( such as administration of antithrombin or recombinant tissue factor pathway inhibitor ) were less successful . further insight into the various mechanisms of action of rhapc on the entangled processes of inflammation and coagulation may permit more detailed dissection of the relative importance of the various pathways that contribute to the pathogenesis of sepsis , potentially culminating in improved treatment strategies . apc = activated protein c ; c4bbp = c4b - binding protein ; dic = disseminated intravascular coagulation ; epcr = endothelial protein c receptor ; par = protease activated receptor ; rhapc = recombinant human activated protein c. ml and tvdp have participated in advisory boards of eli lilly and company and have participated as investigators in experimental and clinical studies with rhapc . this article is part of critical care volume 11 supplement 5 : severe sepsis and drotrecogin alfa ( activated ) .
mononeuritis multiplex involves inflammation of two or more nerves , typically in unrelated parts of the body . it has been well described in bleeding disorders like idiopathic thrombocytopenic purpura ( itp ) and hemophilia . acquired amegakaryocytic thrombocytopenia ( aat ) is a bleeding diathesis characterized by thrombocytopenia but with reduced number of megakaryocytes in the bone marrow , as against itp . though aat is a well described entity , peripheral nervous system manifestations have not been described so far . we report a young man who has presented with bleeding diathesis and mononeuritis multiplex due to aat . the mechanism of development of mononeuritis multiplex and treatment options are discussed .	acquired amegakaryocytic thrombocytopenia ( aat ) is a rare disease characterized by immunologically mediated peripheral thrombocytopenia similar to idiopathic thrombocytopenic purpura ( itp ) . however , in contrast to itp , there is selective reduction / absence of bone marrow megakaryocytes . though multiple simultaneous / sequential peripheral nerve involvement ( mononeuritis multiplex ) is rarely described in itp , it is hardly ever described in aat . we are reporting such a case of mononeuritis multiplex due to a cyclical type of aat in a young man . a 34-year - old man presented in neurology service with pain in the legs , bilateral foot drop and numbness in the medial aspect of the left forearm of 2 weeks duration . examination showed pupura and on positive questioning he admitted bleeding from gums starting 2 days prior to neurological symptoms . at the age of 12 years , he had multiple blood transfusions and was told to have aplastic anemia , but a detailed medical report was not traceable . at the age of 22 years , he had an acute hepatitis b infection when the blood counts and hematocrit were documented to be normal . at the time of index admission , he had anemia and multiple purpuric skin lesions [ figure 1 ] . clinical neurological examination revealed sensory motor involvement of the left radial , left ulnar , right common peroneal and left sciatic nerves suggesting a mononeuritis multiplex . an electrophysiology evaluation showed the sensory motor involvement of the left ulnar , left median , left radial , right median , right common peroneal and left sciatic nerves confirming mononeuritis multiplex . there were conduction blocks demonstrable in right peroneal and left ulnar nerves [ figure 2 ] . after the neurological investigations , he was referred to a government teaching hospital where his subsequent hematological workup was done . echymotic and purpuric skin lesions in legs ( arrows ) motor conduction blocks ( more than 50% reduction in compound muscle action potential amplitude with proximal stimulation compared to distal stimulation ) in ( a ) right peroneal nerve and ( b ) left ulnar nerves his hematocrit was 33% ; total leukocyte count , differential count , and erythrocyte sedimentation rates were normal . the platelet count was grossly reduced to just 5000 cells / cu mm and the bleeding time was prolonged to 9 min . corrected reticulocyte count was 2.9% and peripheral blood smear showed marked thrombocytopenia [ figure 3 ] . the serum bilirubin was marginally elevated to 1.86 mg / dl with the predominant indirect component . both direct and indirect coomb 's tests were negative . measurements of serum sodium , potassium , calcium , phosphorus , glucose , urea , creatinine , proteins , aspartate aminotransferase and alkaline phosphatase levels were normal . serological tests for hiv , hepatitis b , hepatitis c and syphilis were negative . peripheral blood smear ( leishmans stain , 100 ) showing markedly reduced platelets ; compare with normal smear in right panel showing platelets ( arrow ) a bone marrow aspiration study , after relative stabilization , showed normoblastic erythroid / myeloid series , normal lymphoid cells and marked reduction of megakaryocytes [ figure 4 ] . he had profuse bleeding from the site of aspiration with a fall in hematocrit up to 17% and hence a bone marrow trephine biopsy and a nerve biopsy were not attempted . he was initiated on oral prednisolone , and since the condition was not improving , later intravenous immunoglobulin was given for 5 days , and cyclosporine a was added . gradually he stabilized , bleeding stopped and platelet count improved to 60,000 cells / cumm . at the time of hospital discharge at 6 weeks , he had only minimal bilateral foot drop and mild left grip weakness . after 1 week of discharge , he was readmitted with acute onset massive intracerebral hemorrhage , to which he succumbed within few hours . absent megakaryocytes in bone - marrow ( leishmans stain , 10 ) ; compare with megakaryocytes of normal bone - marrow in right panel ( arrow ) we report a case of mononeuritis multiplex due to aat . our patient presented with painful bilateral foot drop along with skin lesions . the clinical and electrophysiological features were suggestive of a mononeuritis multiplex since there was asymmetric involvement of multiple peripheral nerves . skin rashes in mononeuritis multiplex are not rare , and the major differentials include collagen vascular diseases , systemic vasculitides , secondary antiphospholipid antibody syndrome , amyloidosis , sarcoidosis , lyme disease , leprosy and postviral syndromes . hepatitis b virus infection needs special mention since its long - term autoimmune manifestations like systemic lupus erythematosus , antiphospholipid syndrome , polyarteritis nodosa and rheumatoid arthritis can cause mononeutis multiplex with a skin rash . however , hepatitis b serology was negative in our patient though he had an acute infection previously . detailed evaluation revealed markedly reduced megakaryocytes in the bone marrow with relatively normal erythroid and myeloid series . this hematological picture was consistent with what is described as aat , as against itp with similar peripheral blood picture , but normal or increased megakaryocytes in bone marrow . the neuropathy in our patient was probably due to bleeding into the nerves as a result of thrombocytopenia . similar neuropathy is well described in itp , where the cause was histologically proven to be intraneural hematoma beneath the epineurium , in between the fascicles . amegakaryocytic thrombocytopenia is a rare disease characterized by severe thrombocytopenia with a total absence or a selective decrease in bone marrow megakaryocytes . it may be a primary disorder or may be seen in aplastic anemia , preleukemia , systemic lupus erythematosus , congenital rubella , dengue fever , ethanol abuse , nutritional b-12 deficiency , radioiodine therapy and thrombocytopenia - absent radius syndrome . a variety of pathogenetic mechanisms were suggested , but an immune - mediated mechanism , due to an autoantibody that blocks the action of endogenous thrombopoietin on megakaryocytopoiesis , appears sensible , at least in a subset of patients . while some patients remain clinically stable , others may progress to aplastic anemia or myelodysplastic syndrome . a cyclical course has also been described , and our patient appears to be in this category though the hematological picture during the initial episode is not clear . congenital amegakaryocytic thrombocytopenia ( cat ) is another related rare disease presenting with isolated thrombocytopenia in infancy , developing into pancytopenia in later childhood . as against the immune - mediated mechanism in aat , defective surface expression of the thrombopoetin receptor c - mpl over hematopoietic progenitor cells due to c - mpl mutations various treatments have been tried in aat , including corticosteroids , lithium carbonate , androgens , vincristine , folic acid , platelet and erythrocyte transfusions and plasma substitution . an occasional patient may respond well to an intravenous immunoglobulin or cyclosporin a. but , the only real hope is bone - marrow transplantation using a human leukocyte antigen - matched donor .
hypertension , diabetes mellitus ( especially type 2 diabetes mellitus ) , metabolic syndrome and obesity are rapidly growing public health problems . sympathetic nerve activation is observed in obesity , hypertension and diabetes mellitus , which have strong genetic as well as environmental determinants . reduced energy expenditure and resting metabolic rate are predictive of weight gain , and the sympathetic nervous system participates in regulating energy balance through thermogenesis . the thermogenic effects of catecholamines in obesity have been mainly mediated via the 2- and 3-adrenergic receptors in humans . further , 2-adrenoceptors importantly influence vascular reactivity and may regulate blood pressure . genetic polymorphistns of the -adrenoceptor gene have been shown to alter the function of several adrenoceptor subtypes and thus to modify the response to catecholamine . 2-adrenoceptor polymorphisms ( arg16gly , gln27glu , and thr164ile ) have been studied in relation to hypertension . genetic variations in the 3-adrenoceptor ( i.e. try64arg variant ) are also associated with both obesity and hypertension . however , the precise relationships of the polymorphisms of 2- and 3-adrenoceptor genes with sympathetic nervous system activity , hypertension , and metabolic syndrome have not been fully clarified . this paper will discuss the current topics involving the influence of the sympathetic nervous system and 2- and 3- adrenoceptor polymorphisms in hypertension and metabolic syndrome .	obesity , hypertension , and metabolic syndrome ( type 2 diabetes mellitus ) are major and growing health problems and are known as high - risk factors for subsequent cardiovascular and renal complications [ 13 ] . obesity , hypertension , diabetes , and metabolic syndrome are intimately associated [ 46 ] , and sympathetic nervous activation is frequently observed in those conditions . thus , sympathetic nerve activation may play a major role in the onset and development of hypertension , obesity , and metabolic syndrome ( diabetes mellitus ) as well as cardiovascular complications in patients with hypertension , diabetes and obesity [ 2 , 7 ] . the sympathetic nervous system plays an important role in the regulation of energy expenditure . reduced energy expenditure and resting metabolic rate are predictive of weight gain ( obesity ) . a large part of the sympathetic nervous system - mediated energy expenditure takes place in skeletal muscle , via the coupling of catecholamines with 2-adrenoceptors . catecholamines are also powerful regulators of lipolysis and act via 1- , 2- , 3- ( stimulatory ) , and 2- ( inhibitory ) adrenoceptor subtypes in adipose tissue , where their role becomes especially important during both exercise and energy restriction , when increased need for fat as a fuel exists . thus , -adrenoceptors play important roles in energy expenditure and control body weight [ 913 ] . recently , there is evidence that human hypertension and obesity have strong genetic backgrounds [ 1416 ] . reported that about 46% of the phenotype of systolic blood pressure are determined genetically for hypertension [ 17 , 18 ] . [ 1822 ] have reported close relationships between 2- and 3-adrenoceptor polymorphisms accompanying elevated sympathetic nervous activity , blood pressure elevation ( hypertension ) , weight gain ( obesity ) , and insulin resistance in a series of longitudinal study . many epidemiological studies on the relationships between -adrenoceptor polymorphisms , hypertension , obesity , and diabetes ( metabolic syndrome ) have still been discordant . this paper will discuss the current topics involving the contribution of the sympathetic nervous system and 2- and 3-adrenoceptor polymorphisms in the onset and the development of hypertension and metabolic syndrome ( type 2 diabetes mellitus ) . the adrenoceptors ( or adrenergic receptors ) are a class of g protein - coupled receptors which specifically bind their endogenous ligands , the catecholamines ( epinephrine and norepinephrine ) . many tissues possess these adrenoceptors , and the binding of an agonist generally elicits a typical sympathetic response ( i.e. , the fight - or - flight response ) . table 1 shows the effects of catecholamines bound to adrenoceptors ( table 1 ) and these effects on sympathetic nervous activity are through - and -adrenergic receptors . there are several types of adrenergic receptors , but there are two main groups : -adrenoceptors ( 1- and 2-adrenoceptors ) and -adrenoceptors ( 1- , 2- , and 3-adrenoceptors ) . table 1 also summaries the distributions and functions of the 1- , 2- , 1- , 2- , and 3-adrenoceptors [ 24 , 25 ] . phenylephrine is a selective agonist of the -adrenoceptors ( both 1- and 2-receptors ) , thus phenylephrine is usually used to investigate the -adrenoceptors function . downstream effects of cyclic amp include cyclic amp dependent protein kinase , which mediates the intracellular events following hormone binding . insulin resistance in hypertension has been well documented in many epidemiological and clinical studies [ 8 , 26 , 27 ] . several investigators have reported that chronic insulin administration elevates blood pressure in rats and in humans , although insulin also has effects on vasodilation . in addition , many clinical and epidemiological studies have demonstrated the close relationships between sympathetic nerve activity , insulin resistance and hypertension [ 19 , 2932 ] . landsberg and other investigators examined the effect of feeding and starvation on sympathetic nerve activity in the cardiac tissue of animals , noting that feeding raised sympathetic nerve activity , and starvation had the opposite effect [ 3335 ] . energy intake stimulates hyperinsulinemia and sympathetic nerve activity resulting in blood pressure elevations in a cycle to inhibit thermogenesis . insulin - mediated sympathetic nerve stimulation in obese subjects is a compensatory mechanism aimed at restoring the energy balance by increasing the metabolic rate . therefore , hyperinsulinemia and insulin resistance in obese subjects are all part of a response to limit further weight gain via stimulating sympathetic nerve activity and thermogenesis . on the other hand , julius et al . have hypothesized that increased sympathetic nerve activity in skeletal muscle causes neurogenic vasoconstriction , thereby reducing blood flow to muscle and consequently inducing a state of insulin resistance by lowering glucose delivery and uptake in hypertension and obesity . both blood pressure elevation and weight gain may reflect a primary increase in sympathetic nervous tone . they described that high plasma norepinephrine might predict future blood pressure elevations and weight gain accompanying deterioration in insulin resistance observed in homa - ir ( homeostasis model assessments of insulin resistance ) [ 30 , 37 ] . their results suggest that sympathetic nerve activity might play a major role in the development of insulin resistance accompanying blood pressure elevations . reported attenuation of hemodynamic and energy expenditure responses to isoproterenol infusion in hypertensive patients , suggesting that sympathetic nerve activity - induced hypertension may subsequently lead to the development of obesity . many epidemiological studies showed close linkages of beta2- and beta3-adrenoceptor polymorphisms with obesity , hypertension , and the metabolic syndrome shown in tables 2 , 3 , and 4 . thus , close linkages between sympathetic nerve activity and insulin resistance might depend on the -adrenoceptor polymorphisms . thus , one could speculate that the strong associations between -adrenoceptor polymorphisms and insulin resistance might provide evidence that heightened sympathetic nerve activity followed by insulin resistance might play a major role in hypertension and obesity , because -adrenoceptor polymorphisms might relate to insulin resistance through heightened sympathetic nerve activity ( figure 1 ) . the sympathetic nervous system plays an important role in the regulation of energy expenditure and blood pressure regulation . a large part of the sympathetic nervous system - mediated energy expenditure takes place in skeletal muscle , via the coupling of catecholamines with 2-adrenoceptors . catecholamines are also powerful regulators of lipolysis and act via 1- , 2- , 3- ( stimulatory ) , and 2- ( inhibitory ) adrenoceptor subtypes in adipose tissue , where their role becomes especially important during both exercise and energy restriction , when increased need for fat as a fuel exists . stimulation of -adrenergic receptors by the sympathetic nervous system is a significant physiological modulator of pre- and postprandial energy expenditure [ 1113 ] and total daily energy expenditure [ 9 , 10 ] . single nucleotide polymorphisms might have functional consequences in terms of receptor activity and regulation and hence may contribute to the pathophysiology of hypertension and obesity . on the other hand , there are few studies on the relationships between -adrenoceptor polymorphisms , hypertension , obesity , and metabolic syndrome . the 1-adrenoceptor is predominantly expressed in cardiac myocytes and adipose tissue , where its activation leads to increased heart rate and contractility and stimulation of lipolysis , respectively . the two most common 1-adrenoceptor polymorphisms are ser49gly and arg389gly , with relative allele frequencies of 0.85/0.15 and 0.70/0.30 in the caucasian population , respectively . the 1-adrenoceptor is a candidate gene for obesity because of its role in catecholamine - mediated energy homeostasis [ 72 , 73 ] . for example , in obese individuals , the degree of weight loss during a very low calorie diet has been shown to correlate with changes in 1-adrenoceptor protein concentration in adipose tissue . a population cohort of 761 women showed that women carrying the gly49 genotype had greater increases in bmi over15 years compared to those with the ser49 genotype . conversely , the distribution of the arg389gly polymorphism is similar in lean and obese subjects and in a large cohort study including 3981 normotensive and 2518 hypertensive subjects . the factors which might explain the discrepancy of published data are shown in the later section . the 2-adrenoceptor is the dominant lipolytic receptor in white human adipose tissue and in skeletal muscle . genetic polymorphisms of the 2-adrenoceptor have been associated with hypertension , obesity , and metabolic syndrome ( diabetes mellitus ) . the most common polymorphisms are arg16gly , with an allele frequency of 0.40/0.60 , and gln27glu , with an allele frequency of 0.55/0.45 , in the caucasian population . the thr164ile polymorphism is rare , occurring in only 3 to 5% of the general caucasians population . studies of agonist stimulation in cultured cells demonstrate that gly16 receptors have a greater reduction in numbers or enhanced downregulation when compared with arg16 whereas the glu27 receptor is resistant to down regulation when compared with the gln27 variant . a number of clinical studies have investigated the impact of these polymorphisms on vascular responsiveness [ 40 , 109 ] . . found that young normotensive white men homozygous for the gly16 allele had higher blood pressure and lower peripheral vasodilation after infusion of the 2-agonist salbutamol . similar results were obtained by hoit et al . using the agonist terbutaline . on the other hand , three studies investigating isoprenaline induced increase in the limb blood flow thus , volunteers homozygous for gly16 exhibited larger vasodilatory responses than did volunteers homozygous for arg16 . conflicting results have also been published with regard to the effects of genetic variants on the sympathetic nervous system modulation of energy expenditure . reported that the response of resting energy expenditure to nonspecific -adrenoceptor stimulation ( with isoproterenol infusion ) was not different between the 3 genotypes of arg16gly . . showed that individuals carrying the arg16arg variant of the 2-adrenoceptor gene have a reduced thermogenic response to selective 2-adrenoceptor activation . associations of 2-adrenoceptor polymorphisms with hypertension and metabolic syndrome have been reported in many epidemiological studies but results are also discordant ( summarised in tables 2 and 3 ) . the 3-adrenoceptor , which is mainly expressed in adipose tissue , differs from the 2-adrenoceptor in two ways : it has a lower affinity for catecholamines , and it resists desensitisation ( i.e. , downregulation ) . these characteristic differences might lead to the different effects of catecholamine on 2-adrenoceptors and 3-adrenoceptors . 3-adrenoceptors stimulate the mobilization of lipids from the white fat cell and increase thermogenesis in brown fat cell . decreased function of 3-adrenoceptor in white adipose tissue slow lipolysis may contribute strongly to visceral obesity in human , and treatment of obese animal models with selective 3-adrenergic agonists reduces fat stores most effectively [ 94 , 113 , 114 ] . many epidemiological studies have shown the strong relationships between 3-adrenoceptor polymorphisms ( mainly trp54arg ) , hypertension , metabolic syndrome , and obesity [ 78 , 94 , 113117 ] ( table 4 ) . tables 2 , 3 , 4 , and 5 show the discordant contributions of -adrenoceptor polymorphisms to hypertension , metabolic syndrome ( type 2 diabetes ) , and obesity . further , haplotypes of polymorphisms have strong influence on -adrenoceptor function in each polymorphism [ 20 , 58 , 59 , 105107 ] . the role of the sympathetic nervous system 2- and 3-adrenoceptor polymorphisms in hypertension , metabolic syndrome ( diabetes mellitus ) , and obesity is discussed through a literature review . sympathetic nervous system activity and -adrenoceptor polymorphisms ( mainly 2- and 3-adrenoceptor polymorphisms ) might contribute to the onset and maintenance of hypertension , metabolic syndrome , and obesity ; however , the findings have been discordant . further , few studies have been performed to evaluate the relationship between 2- and 3-adrenoceptor polymorphisms and sympathetic nervous system activity in the same study . a better understanding for the relationships of genetic background ( polymorphisms ) with sympathetic nervous system activity as the cause for hypertension ( blood pressure elevation ) , metabolic syndrome ( insulin resistance ) , and obesity ( weight gain ) might help for clinical treatment for obesity - related hypertension and metabolic syndrome . in fact , a number of studies have investigated genetic polymorphisms as determinants of cardiovascular response to antihypertensive drug therapy [ 103 , 104 ] . but in addition , to clarify the pathogenesis and mechanisms may lead to the prevention of hypertension and metabolic syndrome in obesity .
fragile x mental retardation syndrome is a repeat expansion disease caused by expansion of a cggccg - repeat tract in the 5 utr of the fmr1 gene . in humans , small expansions occur more frequently on paternal transmission while large expansions are exclusively maternal in origin . it has been suggested that expansion is the result of aberrant dna replication , repair or recombination . to distinguish amongst these possibilities we crossed mice containing 120 cggccg - repeats in the 5 utr of the mouse fmr1 gene to mice with mutations in atr , a protein important in the cellular response to stalled replication forks and bulky dna lesions . we show here that atr heterozygosity results in increased expansion rates of maternally , but not paternally , transmitted alleles . in addition , age - related somatic expansions occurred in mice of both genders that were not seen in atr wild - type animals . some atr - sensitive expansion occurs in postmitotic cells including haploid gametes suggesting that aberrant dna repair is responsible . our data suggest that two mechanisms of repeat expansion exist that may explain the small and large expansions seen in humans . in addition , our data provide an explanation for the maternal bias of large expansions in humans and the lower incidence of these expansions in mice .	expansion of a tandem repeat array is responsible for disease pathology in the repeat expansion diseases , a group of genetic disorders that includes fragile x mental retardation syndrome ( fxs ) ( 1,2 ) . in fxs however , it differs from the genome - wide microsatellite instability seen in diseases like hereditary non - polyposis colorectal carcinoma ( hnpcc ) in showing an expansion bias ( more expansions than contractions ) and occurring at a single genetic locus . in addition , at least in the case of mouse models for the disorders resulting from cagctg - repeat expansion , mutations in dna mismatch repair genes , like msh2 , msh3 and pms2 , actually decrease the frequency of repeat expansion while the opposite is true for hnpcc ( 39 ) . different diseases in this group involve repeats with different sequences and repeat unit sizes ( 10 ) . these repeats have the potential to form secondary structures that are thought to play a role in the expansion process [ see ( 11 ) for a recent review ] . however , since not all of the repeats have the same properties , it is unclear whether all repeats expand via the same mechanism . studies in bacteria and yeast have shown that variety of mechanisms can cause repeat instability in these organisms including dna slippage during replication , errors in okazaki fragment processing as well as aberrant dna repair or recombination ( 1242 ) . however , much is currently unknown about the events responsible for expansion in humans . for example , some expansion diseases only show small changes in repeat number on intergenerational transfer , while others result in alleles many times larger than the parental allele from which they are derived . the small expansions are typically seen when the repeats fall within an open reading frame , as in the case of the cagctg - repeat responsible for huntington disease ( hd ) . large expansions , like those that cause fxs , are characteristically seen in regions outside of the open reading frame and occur almost exclusively on maternal transmission . in addition , some diseases involve significant somatic instability while others do not and it is not known if the same mechanism is responsible for both germline and somatic expansion . some studies in transgenic mice have suggested that small expansions occur premeiotically in spermatogonia ( 9 ) , whilst others suggest that expansions occur in haploid gametes ( 8,43 ) . some studies also suggest a second event occurs in the early female embryo . in some transgenic mouse models this event is an expansion ( 44 ) and in others it is a contraction ( 45,46 ) . the differences in these two models has been attributed to the different genomic context of the repeats ( 44 ) . this would be consistent with work in bacteria , yeast and tissue culture models that have implicated orientation , proximity to origins of replication and transcription as cis - acting factors affecting expansion ( 15,21,24,27,31,36,38,39,47,48 ) . in order to study the fragile x repeat in as close to normal a chromosomal context as possible , we generated a fxs premutation knock - in ( ki ) mouse containing 120 cggccg - repeats in the murine fmr1 gene ( 49 ) . since the murine fmr1 gene is located in a region of the x chromosome that is syntenic with the corresponding region of the human x chromosome , differences in cis - acting signals involved in expansion may be small . as was seen in other transgenic and knockin mouse models of cggccg - repeat expansion ( 5053 ) , instability in these animals resembles what is seen in humans in that the frequency is high and shows an expansion bias ( 49 ) . in our mouse model large expansions that generate alleles in the full mutation range ( > 200 repeats ) were seen , but at a much lower frequency than in humans ( 49 ) . in fact most expansions in these mice are small , involving fewer than five repeats per generation and occurring more commonly in males than in females . in this respect these expansions resemble what is seen in human carriers of fmr1 common or intermediate sized alleles ( grey - zone alleles ) ( 54 ) or those diseases such as hd that involve relatively small increases in repeat number . expansion occurring during the perigametic interval between the last premeiotic mitosis and the first post - meiotic one could explain the differences between mice and humans and the maternal cggccg - repeat expansion bias in the human fmr1 gene . this interval can last decades in human females creating a large window of opportunity for expansion to occur . in contrast , this interval lasts only months in female mice and in weeks in males of either species . depletion of the dna repair capacity for much of spermatogenesis could also exacerbate the differences in maternal and paternal expansion rates ( 55 ) . it may also be that some of the differences between humans and mice reflect differences in the efficacy of dna damage repair or checkpoint proteins . one potential dna damage checkpoint protein that may affect expansion frequency is the ataxia - telangiectasia and rad3-related ( atr ) kinase . atr primarily responds to stalled replication forks and bulky dna adducts like those arising from uv irradiation ( 56 ) or s(n)1-type alkylating agents ( 57 ) . here we show that atr heterozygosity leads to increased maternally transmitted expansions and somatic expansions in mice of both genders that appear to involve aberrant dna repair . the atr mice were a kind gift of dr eric brown ( caltech , pasadena , ca ) . mice were maintained in accordance with the guidelines of the niddk animal care and use committee and with the guide for the care and use of laboratory animals ( nih publication no . genomic dna is prepared from mouse tail dna or homogenized mouse tissue as previously described ( 49 ) . genotyping to detect the presence or absence of the disrupted atr gene the primer pair , frax - c and frax - f ( 1 ) , was used to detect both wildtype ( wt ) fmr1 and fxs premutation alleles . the size of the cggccg - repeat tract was monitored by polymerase chain reaction ( pcr ) using the primers frax - m4 ( 5-cttgaggcccagccgccgtcggcc-3 ) and frax - m5 ( 5-cggggggcgtgcggtaacggcccaa-3 ) . the binding sites for these primers are located immediately adjacent to the repeat tract and their 3 ends are unique to the ki allele . radiolabelled pcr products were generated by inclusion of -p - dctp in the reaction mix as previously described ( 49 ) . the reaction products were then run on a 3130xl genetic analyzer and analysed using genemapper 3.7 ( applied biosystems , foster city , ca ) . these bands show a gaussian distribution about the mean that is very similar for alleles in the same size range . the mean size of each allele was calculated based on the mobility of the central band in the cluster . comparison of the parental mean and the offspring 's ; mean determined from samples run on the same gel , allows the changes in the offspring 's ; allele to be reproducibly determined . statistical analysis of instability was carried out using the chi square and student 's t - tests . the atr mice were a kind gift of dr eric brown ( caltech , pasadena , ca ) . mice were maintained in accordance with the guidelines of the niddk animal care and use committee and with the guide for the care and use of laboratory animals ( nih publication no . genomic dna is prepared from mouse tail dna or homogenized mouse tissue as previously described ( 49 ) . genotyping to detect the presence or absence of the disrupted atr gene the primer pair , frax - c and frax - f ( 1 ) , was used to detect both wildtype ( wt ) fmr1 and fxs premutation alleles . the size of the cggccg - repeat tract was monitored by polymerase chain reaction ( pcr ) using the primers frax - m4 ( 5-cttgaggcccagccgccgtcggcc-3 ) and frax - m5 ( 5-cggggggcgtgcggtaacggcccaa-3 ) . the binding sites for these primers are located immediately adjacent to the repeat tract and their 3 ends are unique to the ki allele . the pcr reaction was done in one of two ways . radiolabelled pcr products were generated by inclusion of -p - dctp in the reaction mix as previously described ( 49 ) . the reaction products were then run on a 3130xl genetic analyzer and analysed using genemapper 3.7 ( applied biosystems , foster city , ca ) . these bands show a gaussian distribution about the mean that is very similar for alleles in the same size range . the mean size of each allele was calculated based on the mobility of the central band in the cluster . comparison of the parental mean and the offspring 's ; mean determined from samples run on the same gel , allows the changes in the offspring 's ; allele to be reproducibly determined . statistical analysis of instability was carried out using the chi square and student 's t - tests . the checkpoint protein atr is responsible for activating pathways that lead to the repair of stalled dna replication forks and bulky lesions in dna . thus the effect of atr mutations on the expansions seen in fragile x premutation mice may shed light on the mechanism of repeat expansion . however , since atr heterozygous mice show a small increase in tumour incidence and a small decrease in overall survival it is apparent that some effects of atr deficiency can be seen even in the heterozygous state ( 58 ) . we thus analysed the transmission of a fxs premutation allele with 120 cggccg - repeats in mice heterozygous for a disrupted atr gene . the results obtained for the repeat length changes in the offspring of these mice are summarized in table 1 . table 1.expansions of a premutation allele in wt and atr mice on paternal and maternal transmissionoffspringcross% mice with expansionsmean no . addedmale female(1 ) total(2 ) males(3 ) females(4 ) atr(5 ) wt1fmr1 , atr fmr1 , atr37393337 2.22fmr1 , atr fmr1 , atr86898396635.03fmr1 , atr fmr1 , atr6161613.14fmr1 , atr fmr1 , atr686869675.25fmr1 , atr fmr1 , atr636363632.2w : wildtype fmr1 allele ; : fmr1 premutation allele ; - : indicates offspring from crosses that should not have either the premutation or a mutated atr allele ; * : data source : entezam et al . ( 47 ) ; numbers sharing one of these symbols were compared using the chi - squared test and shown to be significantly different with a p - value < 0.005 . numbers sharing these symbols were compared using the student 's t - test and shown to be significantly different with a p - value < 0.005 . expansions of a premutation allele in wt and atr mice on paternal and maternal transmission w : wildtype fmr1 allele ; : fmr1 premutation allele ; - : indicates offspring from crosses that should not have either the premutation or a mutated atr allele ; * : data source : entezam et al . ( 47 ) ; numbers sharing one of these symbols were compared using the chi - squared test and shown to be significantly different with a p - value < 0.005 . numbers sharing these symbols were compared using the student 's t - test and shown to be significantly different with a p - value < 0.005 . atr heterozygosity had no effect on the deletion frequency and no effect of atr heterozygosity was seen on the stability of the normal mouse fmr1 repeat ( data not shown ) . in contrast , a significant increase in the expansion frequency was seen when the premutation allele was maternally transmitted in atr mice compared to wt mice ( 86% versus 37% ; cross 2 versus cross 1 in table 1 ) . this suggests that atr is normally involved in protecting the genome against intergenerational expansions in female mice carrying fxs premutation alleles . in contrast , the expansion frequency on paternal transmission by atr mice was not statistically different from mice wt with respect to atr ( 68% versus 61% , cross 4 versus cross 3 in table 1 ) . there was an apparent increase in the average number of repeats added per expansion on both paternal and maternal transmission in atr heterozygotes despite the fact that no increase in expansion frequency was seen on paternal transfer . there was no significant gender bias in the expansion frequency in the offspring of atr mothers . this is similar to what is seen in with fragile x repeats in humans ( 59 ) and differs from the male expansion bias seen in a transgenic mouse model of cagctg - expansions ( 45 ) . atr wt males showed no increase in the transmission of an expanded cggccg - allele when crossed to atr females ( cross 5 in table 1 ) . an expansion frequency of 63% was seen in the wt progeny of females carrying the premutation who are heterozygous for atr ( cross 2 ) . this is significantly higher than the 37% seen in offspring of females wt for atr . the expansion frequency was even higher in the atr offspring of atr mothers ( 96% , cross 2 in table 1 ) . to study the role of atr in somatic instability we examined the size of the repeat in different organs of young ( 10 weeks old ) and old ( 18 months old ) atr mice . as can be seen from figure 1a , very limited somatic instability was seen in the liver of young mice as evidenced by the very slightly skewed distribution of repeat sizes . old mice showed much more significant changes in organs like brain , testes and liver . in some instances little remained of the original allele size ( see brain sample in figure 1c ) . we have previously shown that no such somatic instability was seen in mice of a similar age that were wt for atr ( 49 ) . in some organs , like the male brain , the expansions resulted in a shift of the average allele size without changing the basic monophasic distribution of allele sizes ( figure 1c ) . in organs like liver and whether the biphasic distribution reflects a predisposition of certain cells within the organ to expand is currently under investigation . a biphasic distribution of somatic expansion products has been reported in the liver in a mouse model knockin mouse model for myotonic dystrophy , a cagctg - expansion disorder , where it was attributed to changes in ploidy in a subset of liver cells ( 60 ) . figure 1.somatic instability in atr mice carrying an fxs premutation allele . genomic dna was isolated from various organs of young ( 10 weeks old ) and old ( 18 months old ) fxs premutation mice and the repeat tract analysed by pcr using one fam - labelled primer and an abi geneanalyzer as described in the materials and methods . the number repeats in the modal allele in the tail dna at 3 weeks of age is shown in black font and is indicated in the other organ samples by the grey dotted line . similar results were obtained using pcr using p--dctp and denaturing gel electrophoresis as described previously ( 49 ) . genomic dna was isolated from various organs of young ( 10 weeks old ) and old ( 18 months old ) fxs premutation mice and the repeat tract analysed by pcr using one fam - labelled primer and an abi geneanalyzer as described in the materials and methods . the number repeats in the modal allele in the tail dna at 3 weeks of age is shown in black font and is indicated in the other organ samples by the grey dotted line . similar results were obtained using pcr using p--dctp and denaturing gel electrophoresis as described previously ( 49 ) . atr heterozygosity had no effect on the deletion frequency and no effect of atr heterozygosity was seen on the stability of the normal mouse fmr1 repeat ( data not shown ) . in contrast , a significant increase in the expansion frequency was seen when the premutation allele was maternally transmitted in atr mice compared to wt mice ( 86% versus 37% ; cross 2 versus cross 1 in table 1 ) . this suggests that atr is normally involved in protecting the genome against intergenerational expansions in female mice carrying fxs premutation alleles . in contrast , the expansion frequency on paternal transmission by atr mice was not statistically different from mice wt with respect to atr ( 68% versus 61% , cross 4 versus cross 3 in table 1 ) . there was an apparent increase in the average number of repeats added per expansion on both paternal and maternal transmission in atr heterozygotes despite the fact that no increase in expansion frequency was seen on paternal transfer . there was no significant gender bias in the expansion frequency in the offspring of atr mothers . this is similar to what is seen in with fragile x repeats in humans ( 59 ) and differs from the male expansion bias seen in a transgenic mouse model of cagctg - expansions ( 45 ) . atr wt males showed no increase in the transmission of an expanded cggccg - allele when crossed to atr females ( cross 5 in table 1 ) . an expansion frequency of 63% was seen in the wt progeny of females carrying the premutation who are heterozygous for atr ( cross 2 ) . this is significantly higher than the 37% seen in offspring of females wt for atr . the expansion frequency was even higher in the atr offspring of atr mothers ( 96% , cross 2 in table 1 ) . to study the role of atr in somatic instability we examined the size of the repeat in different organs of young ( 10 weeks old ) and old ( 18 months old ) atr mice . as can be seen from figure 1a , very limited somatic instability was seen in the liver of young mice as evidenced by the very slightly skewed distribution of repeat sizes . old mice showed much more significant changes in organs like brain , testes and liver . in some instances little remained of the original allele size ( see brain sample in figure 1c ) . we have previously shown that no such somatic instability was seen in mice of a similar age that were wt for atr ( 49 ) . in some organs , like the male brain , the expansions resulted in a shift of the average allele size without changing the basic monophasic distribution of allele sizes ( figure 1c ) . in organs like liver and whether the biphasic distribution reflects a predisposition of certain cells within the organ to expand is currently under investigation . a biphasic distribution of somatic expansion products has been reported in the liver in a mouse model knockin mouse model for myotonic dystrophy , a cagctg - expansion disorder , where it was attributed to changes in ploidy in a subset of liver cells ( 60 ) . figure 1.somatic instability in atr mice carrying an fxs premutation allele . genomic dna was isolated from various organs of young ( 10 weeks old ) and old ( 18 months old ) fxs premutation mice and the repeat tract analysed by pcr using one fam - labelled primer and an abi geneanalyzer as described in the materials and methods . the number repeats in the modal allele in the tail dna at 3 weeks of age is shown in black font and is indicated in the other organ samples by the grey dotted line . similar results were obtained using pcr using p--dctp and denaturing gel electrophoresis as described previously ( 49 ) . genomic dna was isolated from various organs of young ( 10 weeks old ) and old ( 18 months old ) fxs premutation mice and the repeat tract analysed by pcr using one fam - labelled primer and an abi geneanalyzer as described in the materials and methods . the number repeats in the modal allele in the tail dna at 3 weeks of age is shown in black font and is indicated in the other organ samples by the grey dotted line . similar results were obtained using pcr using p--dctp and denaturing gel electrophoresis as described previously ( 49 ) . we have shown that a maternal atr insufficiency leads to an increase in the frequency of intergenerational expansions . atr heterozygosity also causes the appearance of age - related expansion products in certain organs of older males and females . a number of lines of evidence support a prezygotic origin for the atr - sensitive intergenerational expansions seen in females . the elevated expansion frequency in atr wt offspring of atr heterozygous mothers ( cross 2 , column 5 in table 1 compared to cross 1 , column 5 ) demonstrates that expansion can occur prior to fertilization of the oocyte . this is supported by the fact that the paternal expansion frequency in mice wt for atr is the same whether the dam is atr or wt for atr ( cross 5 in table 1 ) . despite the maternal expansion bias , this together with the fact that female mice do not show more somatic instability in adult tissues than males argues against somatic expansions that occur specifically either in the early female embryo or in the female germline prior to meiosis . we have previously shown that male mice show no bias against the transmission of large repeats ( 49 ) . thus the female expansion bias in an atr background is not likely to be due to somatic expansions that are selected against in males . furthermore , the excess of expansions in atr offspring ( 96% compared to 63% in atr wt offspring ) is not consistent with a somatic origin in the mother either since all her somatic cells would be atr . thus the most likely origin of the additional expansions in the atr offspring is in the haploid oocyte . given that scheduled dna replication does not occur in haploid gametes , dna damage is thus likely to be responsible . the haploid gamete that gives rise to atr wt offspring would not be atr deficient . thus if expansion were confined to this stage , we would not expect the expansion frequency to be higher than that seen in offspring of a mother wt for atr . therefore , some atr - sensitive expansions in the wt offspring of atr mothers could have occurred in the oogonia or diploid oocyte . thus the window of opportunity for expansions in these cells is much larger than it is for haploid gametes although the rate of expansion may in fact be lower . it may be that the atr heterozygosity in these mice reveals the existence of two different intergenerational expansion mechanisms , the first showing a higher expansion frequency in males that is less sensitive to atr haploinsufficiency and the second , occurring predominantly in females , that is sensitive . this is not to say that atr mutations are necessary for maternal expansions in humans , but rather that an atr insufficiency in mice allows events that would have years to accumulate in humans , to be visible within the rodent lifespan . the relatively atr - insensitive mechanism may account for the paternal transmission bias seen with intermediate and grey - zone fmr1 alleles in humans ( 54 ) . a higher mutation frequency in males is usually attributed to errors occurring during dna replication since mature sperm are the product of more rounds of cell division than ova ( 61 ) . while it is possible that the increase in the average expansion size seen on paternal transmission reflects measurement errors , it may be that in males the atr - deficiency simply delays the resolution of the replication problem . for example , in a strand - slippage scenario , such a delay could result in the incorporation of additional bases into the expanded allele without affecting the frequency with the initiation of strand - slippage occurs . atr - sensitive expansions may be more common in female mice since our data suggest that the mechanism responsible is related to the repair of dna damage and not genomic replication . expansions can thus occur at any point during gametogenesis , a process that lasts significantly longer in females . it is appealing to think of this mechanism as the basis for the strong maternal bias in the transmission of fragile x full mutation alleles in humans . since gametogenesis takes so much longer in human females , this may provide a much larger window of opportunity for such expansions to occur even in the presence of normal amounts of atr . one organ that showed evidence of significant somatic expansion is the brain . in some cases very little of the original allele was seen ( see adult male brain in figure 1c ) . since a significant fraction of cells in the adult brain are post - mitotic , somatic expansion is also probably not limited to dividing cells . thus these expansions may also arise from an aberrant dna repair process rather than a problem with scheduled dna replication . expansion limited to some organs could be explained by differences in either the frequency with which the dna damage that initiates expansion occurs in these organs or the frequency with which such mutations are repaired or eliminated . organs such as liver and brain may be predisposed to expansion since atr is expressed at a lower level in cells with a low proliferative capacity ( 62 ) and shows a lower affinity for chromatin in such cells ( 63 ) . in a transgenic mouse model of cagctg - repeat expansion a deficiency of ogg1 , an enzyme involved in the repair of the oxidation product of guanine , 7,8-dihydro-8-oxoguanine , reduces somatic expansion frequency ( 64 ) . oxidative dna damage - induced expansion is likely to be high in organs like brain that in the fragile x premutation mice show elevated levels of atr - sensitive somatic mutations . however , since an ogg1 deficiency did not affect germline instability in the cag cag - mouse model , its significance for intergenerational instability in the cagctg - expansion diseases and in the etiology of fragile x syndrome is unclear . in fragile x premutation mice , an atr insufficiency may prevent error - free dna repair pathways from being activated to repair dna damage , forcing the cell to use a secondary repair pathway that results in expansions . potential pathways could be non - homologous end - joining or some sort of homologous recombination . the effect of atr mutations on the cggccg - expansion frequency in female mice raises the possibility that dna damage checkpoint proteins or proteins involved in dna repair have the potential to affect expansion risk in humans . such transacting factors may explain why the risk of expansion in premutation carriers is lower in those carriers identified by general prenatal screening than in carriers from known fragile x families ( 65 ) . it could also explain why some intergenerational instability is apparent in some families with alleles in the grey zone and not others ( 66 ) and the transition from an allele in the normal size range to a full mutation in two generations reported in one family ( 67 ) . our data also raise the possibility that there may be tissue variation in repeat lengths in older human premutation carriers that may be relevant for diagnosis and the severity of disease symptoms .
a 35-year - old woman was determined to have an ovarian cyst and underwent a right ovarian cystectomy at 10 weeks of gestation . a histopathological examination revealed follicular carcinoma arising in a teratoma . no evidence of metastasis was found after delivery . she underwent a total thyroidectomy , followed by radioactive iodine ( rai ) therapy . however , her serum thyroglobulin level increased to 1,437 ng / ml ( normal range : 0 - 52 ng / ml ) after 10 months . radioiodine scintigraphy and abdominal computed tomography revealed liver metastasis and peritoneal seeding . she underwent debulking surgery of the liver , right salpinx , and peritoneal seeding nodules . a pathological examination showed metastatic follicular carcinoma with focal poorly differentiated features . adjuvant rai therapy was restarted , and her serum thyroglobulin levels returned to normal . in conclusion , metastatic lesions were successfully treated with a combination of debulking surgery and rai therapy . close medical follow - up monitoring serum thyroglobulin levels is mandatory in such patients .	struma ovarii is a rare monodermal ovarian teratoma composed predominantly of thyroid tissue.1 approximately 15% of all ovarian teratomas have a small non - significant focus of thyroid tissue . metastases of malignant struma ovarii occur in < 5% of cases,23 and frequent metastatic sites are the liver , peritoneum , lungs , and bone . we present a case of metastatic follicular carcinoma of the liver and peritoneum arising from struma ovarii . a right ovarian cyst was discovered in a 35-year - old woman during a routine follow - up examination for her second pregnancy . she underwent a right ovarian cystectomy and incidental appendectomy at 10 weeks of gestation . a histopathological examination revealed follicular carcinoma arising in a teratoma with infiltrative growth and lymphovascular invasion . after delivery in october 2010 , she was transferred to our hospital for further evaluation . because there was no definite abnormal hypermetabolic lesion on positron emission tomography ( pet ) , she was followed with regular thyroid function tests . however , her serum thyroglobulin level increased to 1,437 ng / ml ( normal range : 0 - 52 ng / ml ) . she underwent an endoscopic total thyroidectomy in january 2012 , and the histopathological examination revealed incidental nodular hyperplasia in the thyroid gland . laboratory tests , including blood cell counts and serum levels of liver enzymes , electrolytes , and creatinine were within normal limits . i-131 scans revealed increased uptake in the right upper quadrant of the abdomen ( fig . 1a ) . abdominal computed tomography ( ct ) revealed an 8.56.3 cm - sized metastatic mass in the segment 6 of the liver and variable - sized seeding nodules in both the paracolic gutter and the pelvic cavity ( fig . 1b ) , but no systemic metastatic lesions were observed on pet scans ( fig . the liver had an exophytic mass and a demarcation line along segment 6 , and the pelvic cavity showed multiple seeding nodules in the right salpinx and ovary . variably sized seeding nodules were discovered in the right diaphragm , small bowel mesentery , and pelvic cavity . we resected the metastatic lesions in segment 6 of the liver and the right salpinx with the infundibulopelvic ligament . the patient started a soft diet on postoperative day 3 , and the percutaneous drainage tube was removed on postoperative day 5 . the histopathological examination reported that the liver mass and the nodules in the right salpinx and right diaphragm were compatible with metastatic follicular carcinoma ( fig . in particular , the pathologist mentioned that the metastatic lesion of the liver focally showed a poorly differentiated area . she has been subsequently treated with rai therapy and recent serum thyroglobulin levels were 12 ng / ml . von kalden first described struma ovarii in 1895 , which is an ovarian teratoma that contains > 50% mature thyroid tissue . most patients are asymptomatic and have pelvic masses ( 45% ) on screening tests , such as ultrasonography or ct . patients present with acute pelvic pain ( 40% ) , menstrual irregularities ( 9% ) , and hyperthyroidism ( 5 - 8%).14 a preoperative diagnosis of struma ovarii is impossible , but patients with hyperthyroidisim can be diagnosed by measuring serum thyroid - stimulating hormone , and thyroxine , and conducting thyroglobulin i-123 scintigraphy . in contrast , garg et al.3 raised the possibility of a link between pregnancy and struma ovarii , as in the present case . no consensus of opinion exists for treating a thyroid - type carcinoma arising in struma ovarii . primary surgical resection is essential for successful treatment , and various procedures have been reported in the literature ( table 1 ) . a total abdominal hysterectomy and bilateral salphingo - oopherectomy with omentectomy are suitable for postmenopausal women or premenopausal women who have completed childbearing . some investigators have demonstrated that rai therapy reduces recurrence after a total thyroidectomy in cases of metastatic struma ovarii with increased serum thyroglobulin levels . thyroidectomy is useful for excluding primary thyroid cancers with subsequent ovarian metastases and for evaluating the effectiveness of rai therapy.56 adverse effects of rai therapy treatment are transient amenorrhea and premature menopause . studies of pregnancy outcomes after rai therapy reveal no harmful effects ; however , miscarriage may occur when conception occurs within 6 months of the last rai therapy.6 other adjuvant treatments after total thyroidectomy such as chemotherapy and thyroid suppression are effective for treating metastatic struma ovarii.7 serum thyroglobulin levels can be used as a marker of metastatic disease . in contrast , patients with increased serum thyroglobulin levels require a total thyroidectomy followed by rai therapy . in addition , radioiodine imaging can be used as a tool for detecting metastatic lesions.36 in this case report , the patient underwent rai therapy after total thyroidectomy , but serum thyroglobulin levels increased , indicating that rai therapy may have been ineffective for treating her disease , or that the tumour growth was too fast to respond to rai therapy . fortunately , her serum thyroglobulin level returned to the normal range after the second debulking operation . therefore , regular serum thyroglobulin measurements might be one of the surveillance tools for cases such as this . most cases of struma ovarii are benign , but malignant changes have been reported in 5 - 15% of cases.24 extraovarian spread is rare even in malignant struma ovarii and occurs in < 5% of patients.23 papillary carcinoma ( 21% ) and follicular carcinoma ( 54% ) are the most frequent types of carcinomas among thyroid - type carcinomas of struma ovarii.1 a typical follicular carcinoma is more likely to metastasize to the lungs , liver , and central nervous system , whereas a papillary carcinoma tends to involve the abdominal cavity and lymph nodes.8 poor prognostic factors are initial extraovarian spread , adhesion to adjacent organs , size > 5 cm , and > 50% proliferating thyroid tissue.9 our patient developed peritoneal dissemination and huge liver metastases 2 years after the ovarian cystectomy . moreover , the last pathological examination showed that the metastatic follicular carcinoma of the liver had the potential for transformation to poorly differentiated carcinoma compared to that in the initial pathological examination . therefore , this patient required close medical observation with regular thyroglobulin measurements . in conclusion , we present a rare case of metastatic follicular carcinoma to the liver and peritoneum initially arising in struma ovarii of a young woman . these metastatic lesions were successfully treated with debulking surgery and rai therapy after a total thyroidectomy . however , because she had several poor prognostic factors , close medical follow - up by serum thymoglobulin monitoring was mandatory .
proteomic approaches are continuing to make headways in cancer research by helping to elucidate complex signaling networks that underlie tumorigenesis and disease progression . this review describes recent advances made in the proteomic discovery of drug targets for therapeutic development . a variety of technical and methodological advances are overviewed with a critical assessment of challenges and potentials . a number of potential drug targets , such as baculoviral inhibitor of apoptosis protein repeat - containing protein 6 , macrophage inhibitory cytokine 1 , phosphoglycerate mutase 1 , prohibitin 1 , fascin , and pyruvate kinase isozyme 2 were identified in the proteomic analysis of drug - resistant cancer cells , drug action , and differential disease state tissues . future directions for proteomics - based target identification and validation to be more translation efficient are also discussed .	the use of proteomics as a systems biology tool in cancer research continues to expand in scope and depth , as it evolves rapidly into a universally applicable method for the investigation of practically any biological process . proteomics is particularly attractive to cancer research because the complexity of tumorigenesis , cancer progression , tumor relapse , and metastasis often involves large protein networks . indeed , in a little over a decade , applications of mass spectrometry - based proteomics in cancer - related research ranging from mechanistic investigation to the discovery of novel therapeutic targets have increased exponentially . this is evidenced by the explosive growth in the number of publications on the subject matter , from 109 in 2000 to 1,349 in 2011 ( http://www.ncbi.nlm.nih.gov/pubmed ) , with the total number of publications containing the keywords proteomics and cancer reaching over 7,500 to date . a large number of review articles have appeared in the past several years , offering excellent overviews and perspectives on novel proteomic applications in cancer . many reviews focused on different cancer types , such as breast cancer,14 pancreatic cancer,5,6 ovarian cancer,79 colorectal cancer,10,11 and glioma.1214 others have focused on sample types or subcellular components , such as tissue,1517 serum,1820 and secretome.2123 in addition , other reviews focused on the innovative proteomic methodologies or the various end goals of proteomics in cancer , such as phosphoproteomics,2427 mechanisms of drug resistance,2830 and kinome profiling.3133 finally , there appear to be more reviews on proteomic biomarker discovery for cancer diagnosis , prognosis , and therapeutic response than on any other area of focus , highlighting the intense activities in the search for new biomarkers that could be translated to clinical applications.30,3436 despite the remarkable advances in utilizing proteomics in almost every aspect of cancer research , many challenges remain . global proteomic approaches , while capable of identifying thousands of proteins ( many of which are differentially expressed ) are often biased towards soluble and high - abundance proteins , thus easily missing out on many low - abundance proteins that could play critical roles in signal transduction.37 most solid tumors are heterogeneous , making it difficult to identify key proteins that could be used as universal targets even in the same type of cancer.38,39 while high - throughput proteomic methods can discover unbiased cohorts of potential biomarkers and therapeutic targets that regulate disease progression , the sheer number ( often in the dozens , if not the hundreds ) of hits could be cumbersome , as current functional studies are limited to involving one or a few proteins at a time . thus , a large number of diagnostic and prognostic biomarkers have been identified by proteomic analysis , but very few have made it to the clinical stage , underscoring the urgent need for the convergence of functional studies and clinical sample validations . moreover , the progress in drug development has far lagged the pace of proteomic discovery of potential drug targets , as the former generally requires a prolonged developmental process from drug design to in vitro and in vivo tests for efficacy and toxicities , as well as for absorption , distribution , metabolism , and excretion ( adme).40 proteomic approaches have been increasingly used in the preclinical drug discovery process to understand the effects of drug candidates on their protein targets and to shed light on the cellular mechanisms behind the observed phenotype , although many other biomaterials including deoxyribonucleic acid ( dna ) , ribonucleic acid ( rna ) , and carbohydrates can also be used as novel drug targets with different approaches.41 proteomic studies with the main goal of identifying new druggable targets for the treatment of cancer have been relatively few and are the primary focus of this review . for the convenience of discussion , we will attempt to group various proteomic target discovery studies into several distinct approaches for target identification . while mass spectrometry ( ms)-based proteomic approaches remain the most comprehensive and versatile tool in large - scale proteomic profiling,42,43 several non - ms - based techniques , such as reverse phase protein microarrays ( rpma)44 and peptide arrays45,46 have recently gained popularity . rpma is a technology platform designed for the quantitative analysis of specific phosphorylated , cleaved , or total ( phosphorylated and nonphosphorylated ) forms of cellular proteins from complex mixtures in multiple samples simultaneously . one microarray can accommodate a range of hundreds to thousands of samples that are printed in a series of replication . rpma is performed using either a primary or a secondary labeled antibody by chemiluminescent , fluorescent , or colorimetric assays . multiplexing is also achieved by simultaneously probing multiple arrays spotted with the same lysate with different antibodies and can be implemented as a quantitative calibrated assay.47 rpma has now been utilized for potential drug discovery / validation , as well as for advancements in understanding the disease both in the clinic and in the laboratory,48,49 and rpma is currently being integrated into human clinical cancer trials.50 the quantitative methods adopted in ms include techniques based on the stable isotope labeling of proteins / peptides , as well as label - free methods . in comparison with labeling methods , label - free methods provide higher dynamic ranges of quantification and are versatile tools that are used to estimate changes in protein abundances between different samples.5153 the utility of ms - based proteomic analyses and their applications in drug target identification have been increasingly recognized over the past decade due to their high sensitivity , specificity , and throughput . for example , gel - free isotopic labeling methods , such as silac ( stable isotope labeling with amino acids in cell culture),54,55 isobaric tags for relative and absolute quantitation ( itraq),5658 and isobaric tandem mass tags ( tmts)59 ( figure 1 ) can now routinely quantify several hundreds of ( to a few thousand ) proteins in a single analysis with high reproducibility . continued innovations and improvements in the instrumentation and bioinformatics tools will drive ms - based proteomics applications in drug discovery,60,61 complemented by streamlined and focused non - ms - based methods for targeted and repeated assays , such as protein arrays . the former will play key roles in the discovery stage , where quantitative proteomic analysis is undertaken , which compares differentially expressed proteins for potential drug targets . the array methods will be more cost effective and convenient to use for the validation of studies on drug targets and their effects . one of the greatest challenges in cancer therapy is drug resistance that occurs either intrinsically or is acquired after a certain period of treatment . resistance to targeted cancer drugs involves complex and diverse molecular adaptations by cancer cells under the selective pressure of therapeutic regimens . for example , the specific target protein in tumors may undergo mutations to become inaccessible to drugs.6264 in addition , a subset of cancer stem cells may be resistant to the cytotoxicity of drugs that target the bulk of more differentiated cancer cells.6568 moreover , chemotherapeutic agents often induce adaptive changes in the regulatory networks and signaling pathways , leading to the independence of cancer cells on the targeted protein and to the emergence of drug - resistant disease clones.69 indeed , such diverse mechanisms of drug resistance can transform cancer cells under the selective pressure of therapies , which made it extremely difficult to combat tumor recurrence ; this is inevitably followed by metastatic progression of the disease and death . current cancer treatment typically consists of a monotherapy regimen targeting a specific protein or signaling pathway , ( for example , selective estrogen receptor modulators [ serms ] for the estrogen receptor , herceptin for her2 , and gefitinib for epidermal growth factor receptor [ egfr ] ) . however , tumors that are initially responsive eventually develop resistance to therapy via a variety of mechanisms . switching to alternative drugs after the emergence of resistant disease clones following first - line treatment often achieves little in delaying cancer progression . resistance can develop to just one drug , or tumors may be cross - resistant to several structurally dissimilar and functionally distinct agents , a phenomenon known as multidrug resistance.70 thus , there is an urgent need to better understand the resistance mechanism and to identify novel targets for a therapeutic intervention that can be more effective in blocking the progression of cancer cells already resistant to the previous treatment . here , proteomic approaches hold the promise to identify individual proteins and interactive signaling networks that act individually or collectively in conferring drug resistance . the comparison of protein expression between drug - resistant tumor tissues or cell lines with drug - sensitive tumor tissues or cell lines is one of the most promising tools for drug target identification . targeting these proteins and signaling pathways may either resensitize tumor response to the original treatment , or it may help overcoming acquired drug resistance by blocking alternative survival signaling induced by the initial treatment . more importantly , proteomic characterization of a resistant phenotype can provide a more complete picture of signaling adaptation , as multiple drug targets for combinatorial therapeutic solutions can be identified to combat resistance and achieve longer - term improvement in disease outcomes . while advances in this application have been limited , the following examples demonstrate that proteomic approaches are inherently suitable for the discovery of targets for resistant phenotypes , and we expect an increasing number of protein targets to be validated by functional studies followed by therapeutic development . one example comes from a comparative proteomic analysis of colon cancer stem cells and differentiated tumor cells , which uncovered a prominently upregulated protein , baculoviral inhibitor of apoptosis protein ( iap ) repeat - containing protein 6 ( birc6 ) , one of the iaps that may play a crucial role in the chemoresistance of colon cancer stem cells . subsequent knockdown of the gene resulted in resensitization of the cells to chemotherapy.71 results from this proteomic study suggest that birc6 could be used as a potential therapeutic target to eradicate colon cancer stem cells contributing to colon cancer recurrence . in other cases , an itraq ms - based proteomics analysis was used to identify proteins associated with the development of docetaxel resistance by comparing docetaxel - sensitive pc3 cells and docetaxel - resistant pc3-rx cells developed by docetaxel dose escalation.56 functional validation experiments were performed using recombinant protein treatment and small interfering ( si)rna knockdown experiments . the findings from this study suggested that macrophage inhibitory cytokine 1 should be further investigated as a potential biomarker and therapeutic target for acquired resistance to docetaxel treatment . however , extensive functional studies and follow - up validations are required before these proteins can be proven as viable drug targets . similarly , a quantitative proteomic investigation of hepatocellular carcinoma , a type of liver cancer known for its resistance to chemotherapy , implicated phospho - glycerate mutase 1 ( pgam1 ) as a potential therapeutic target.72 clinicopathological analysis indicated that the overexpression of pgam1 was associated with 66.7% of hepatocellular carcinomas , and is strongly correlated with poor differentiation and decreased survival rates ( p<0.01 ) . in addition , short hairpin ( sh)rna - mediated repression of pgam1 expression resulted in significant inhibition in liver cancer cell growth both in vitro and in vivo . using a proteomics approach , elevated levels of prohibitin 1 ( phb1 ) and glutathione s - transferase pi ( gst ) were found to be associated with paclitaxel resistance in discrete subcellular fractions of two drug - resistant sublines relative to their sensitive sublines.73 immunofluorescent staining and fractionation studies revealed an increased level of phb1 on the surface of resistant cell lines . transiently silencing either phb1 or gst gene expression using sirna in the paclitaxel - resistant cancer cell sublines partially sensitized these cells toward paclitaxel . intriguingly , silencing phb1 , but not gst , resulted in the activation of intrinsic apoptosis pathway in response to paclitaxel . similarly , stably silencing either phb1 or gst significantly improved paclitaxel sensitivity in a549tr cells both in vitro and in vivo . this study suggests that phb1 is a mediator of paclitaxel resistance , and this resistance may depend on the cellular localization of proteins . based on these preliminary functional studies , phb1 is proposed as a potential target for therapeutic strategies for the treatment of drug - resistant tumors . again , we have yet to see follow - up investigations of the feasibility of such novel , yet unproven , therapeutic targets . in our recent proteomic study of letrozole - resistant breast cancer , a tmt label was used for a quantitative comparison of protein expression profiles of the resistant versus sensitive mcf-7 cells overexpressing aromatase.74 this study identified fascin , among other significantly upregulated proteins , as a promising therapeutic target for the inhibition of metastasis of hormone - resistant breast cancer cells that have acquired the enhanced capacity of migration and invasion . retrospective clinical validation confirms fascin overexpression as an independent indicator of decreased survival and poor prognosis . a subsequent drug discovery effort by our group led to the discovery of a series of thiazole compounds75 as potent inhibitors of migration and invasion of metastatic cancer cells by binding to fascin . in summary , a resistant cancer population , whether inherent or acquired , is a major problem that reduces the activity of conventional and/or molecularly - targeted cancer drugs . the success of identifying drug targets in resistant cancer cells by proteomic approaches depends on reliable drug - resistant tumor tissues or cell lines . it is also critical that proteomics be combined with data from other approaches in an attempt to investigate mechanistic pathways in more detail , as well as to validate potential targets in a clinical setting . chemical proteomics is a technique that identifies proteins enriched or isolated as a result of interacting with or binding to a chemical probe , usually a small molecule drug that is fixed on a solid support.7678 therefore , these affinity - based enrichment techniques , in combination with ms , have enabled the direct determination of protein - binding profiles of small molecule drugs under physiological conditions and represent one of the most direct approaches to screen for drug protein interactions.79,80 the major drawback encountered in the affinity - based chemical proteomics is the presence in the pulled - down extract of nonspecifically bound proteins . among these , a large number of proteins that bind nonspecifically to most conventional affinity matrices have been reported.81 it is , therefore , strongly recommended that green fluorescent protein be used as the tag of choice because it shows minimal nonspecific binding to mammalian cell proteins , it can be quantitatively depleted from cell extracts , and it allows for the integration of biochemical protein interaction data with in vivo measurements using fluorescence microscopy . in addition , it is also necessary to perform appropriate negative control experiments to distinguish nonspecific interactions from specific interactions . chemical proteomic approaches also include global proteomic profiling of cellular samples treated with a biologically active compound without enrichment steps . in pharmacology , chemical proteomics have been utilized to determine the specificity of drugs and their analogs , for anticipated as well as unknown targets that may also bind to the probe.41 these activity - based probes can specifically target diverse sets of enzyme families and provides direct information about the activation state of identified proteins.82 several kinds of chemical probes have been used in proteomic studies across a multitude of enzyme classes such as hydrolases , proteases , kinases , phosphatases , histone deacetylases , glycosidases , and oxidoreductases.83 thus , chemical proteomic approaches not only identify protein targets for drugs that exert known biological activities in vitro or in vivo , but they also can discover previously unknown targets for drugs of known modes of action ( moa ) . these off - target proteins may be additional druggable targets , but they may also account for the side effects and toxicities . identification of protein targets that are responsible for toxicities may prove valuable in early drug development to minimize failure in clinical trials . in addition , chemical proteomics can identify novel drug targets by broad - action chemical probes ( for example , nonselective kinase inhibitors such as staurosporine [ life technologies , carlsbad , ca , usa]).82 in the following , we present a few examples in which chemical proteomics were used effectively for target discovery and validation . gefitinib ( iressa ; astrazenca , london , uk ) is the first selective inhibitor of the egfr tyrosine kinase domain.84 gefitinib used as monotherapy is an effective treatment for patients with locally advanced or metastatic nonsmall cell lung cancer ( nsclc ) with egfr mutations.85 gefitinib has also been tested in clinical trials in other tumors , including head and neck squamous cell carcinoma ( hnscc ) , as monotherapy , or in combination with other chemotherapies or radiation , but it has shown limited clinical efficacy with response rates of 10%15%.8689 using cleavable isotope - coded affinity tagging ( cicat)-based liquid chromatography tandem mass spectrometry ( lc ms / ms ) method , chen et al90 identified the tyrosine phosphorylation levels of 21 proteins between control and epidermal growth factor - treated a431 human cervical cancer cells . of these , endofin , dcbld2 , and kiaa0582 were validated to be novel tyrosine - phosphorylation targets of epidermal growth factor signaling and responsive to gefitinib therapy.90 pernas et al91 observed that gefitinib sensitivity correlated with phospho ( p)-akt and p - stat3 activation in hnscc cell lines and tumor specimens , thus p - akt and p - stat3 could serve as potentially useful drug targets for further development of novel therapeutic agents for hnscc . more recently , wu et al92 used quantitative chemical proteomics to identify several kinases including nek9 , lyn , jak1 , wee1 , and epha2 , which are involved in cell survival and the proliferation of hnscc cell lines . these findings may lead to new therapeutic options for hnscc patients . for inhibitors acting on multiple tyrosine kinases , chemical proteomic studies could help uncover additional unknown targets and their interacting signaling network . for example , as an inhibitor of multiple tyrosine kinases , dasatinib targets bcr - abl , src family kinases , c - kit , and platelet - derived growth factor receptor kinase , a cell - permeable kinase probe ( da-2 ) was employed to profile potential cellular targets of dasatinib ; a number of previously unknown dasatinib targets were identified , including several serine / threonine kinases ( pctk3 , stk25 , eif-2a , pim-3 , pka c- , and pkn2).93 according to another report,94 nearly 40 different kinase targets of dasatinib were discovered using quantitative phosphoproteomics . these include receptor tyrosine kinases ( ephrin receptors , discoidin domain receptor 1 , and egfr ) and nonreceptor tyrosine kinases ( frk , brk , and ack ) . these results provided a system - level view of dasatinib action in cancer cells and suggested functional targets and rationales for therapeutic strategies . when the mechanisms of biologically active compounds against cancer cells are not fully understood , chemical proteomics could help elucidate the modes of action by mapping out protein networks perturbed by the drug treatment . in this aspect , a wealth of proteomic discoveries have seldom been translated into validated drug mechanisms , most of which stayed as preliminary findings that could lead to confirmed targets by in - depth functional studies . for example , celecoxib ( celebrex ; pfizer , inc , new york , ny , usa ) , originally a widely prescribed nonsteroidal anti - inflammatory drug , has recently been shown to have anticancer properties95,96 with uncertain mechanisms , ranging from modulating the expression of bcl-2 family members and mitochondria - mediated apoptosis,95,97 to inhibiting nuclear factor kappa b,98,99 akt,100 and stat3101signaling pathways . proteomic analysis of human oral squamous cell carcinoma found that celecoxib treatment induced ten- to 20-fold overexpression of heterogeneous nuclear ribonuclear protein c.102 similarly , global proteomic profiles of colorectal cancer cells before and after treatment with celecoxib revealed significant alterations among multiple proteins involved in diverse cellular functions ranging from glycolysis , protein biosynthesis , dna synthesis , messenger rna processing , protein folding , phosphorylation , redox regulation , to molecular chaperon activities.103 however , none of the proteomic studies has been conclusive in the mechanistic interpretation of numerous alterations in protein expression . the wnt--catenin signaling pathway is a developmental signaling pathway that plays a critical role in regulation , differentiation , proliferation , and apoptosis ; thus , aberrant wnt--catenin signaling is widely implicated in numerous cancers.104106 however , identification of targeted wnt--catenin pathway inhibitors in cancer patient treatment has been hampered by the limited number of pathway components that are amenable to small molecule inhibition . recently , huang et al107 used an itraq approach to identify a small molecule , xav939 , that selectively inhibits -catenin - mediated transcription . interestingly , xav939 also inhibits the poly - adenosine diphosphate - ribosylating enzymes , tankyrase 1 and tankyrase 2 , which interact with a highly conserved domain of axin and stimulate its degradation through the ubiquitin proteasome pathway.107 tankyrases , involved in fundamental cellular processes such as telomere homeostasis and wnt signaling , are potential telomere - directed anticancer targets.108,109 therefore , xav939 is not only a useful tool in understanding wnt--catenin signaling , but also a potential drug that targets wnt--catenin and telomeres . chemical proteomics aimed at characterizing the effects of drug candidates could sometimes lead to the discovery of new drug targets . for example , sulforaphane ( sfn ) is known to have antimicrobial and anticancer properties in experimental models.110112 sfn can modulate multiple cellular targets involved in cancer development including dna protection , inhibition of cancer cell proliferation , induction of apoptosis , inhibition of neoangiogenesis , progression of benign tumors to malignant tumors , and metastasis.112,113 mastrangelo et al114 identified serotonin receptors as the novel targets of sfn by proteomic analysis in caco-2 colon cancer cells . this finding may not only shed light on the serotonin - mediated signaling pathways in colon cancer , but it may also lead to the development of potential novel therapeutic agents targeting serotonin . in another study , phosphoglucomutase 3 was identified by proteomic analysis and may contribute to sfn - induced cell death in the lncap prostate cancer ( pca ) cells , which make phosphoglucomutase 3 a potential molecular therapeutic target for pca.115 while these are very early studies on the discovery of druggable protein targets in the relevant disease , they do open up new venues for the development of novel treatment regimens . although the field of chemical proteomics has proven its value in identifying novel drug targets , several challenges still remain to be overcome . first , the chemical probes must be carefully designed to covalently attach to proteins of interest and allow purification and/or identification . second , the spectrum of available activity - based probes needs to be broadened in order to target additional enzyme classes . in addition , the development of high - throughput and gel - free assays , in conjunction with activity - based probes , will be required to enhance the experimental value of chemical proteomics . the continued success of chemical proteomics depends on the design of novel probes or new probe classes that can specifically target diverse sets of enzyme families , as well as on an unbiased assessment of the full spectrum of drug target interactions and their molecular moa . proteins and their spatiotemporal distribution changes play a central role in biological processes , including cancer initiation and progression . analysis of a specific homogeneous cell type from tumor tissues could reveal molecular changes that take place in tumorigenesis since the concentration of disease - related proteins are likely to be much higher within or near the tumor areas . recently , large - scale protein identification and comparative quantitation of highly complex protein mixtures have been achieved with proteomic strategies based upon differential disease state tissues , known as tissue proteomics.35,116120 remarkable advances in tissue proteomics have been propelled by the rapid development of efficient methodologies and techniques in innovative sample preparation , sophisticated ms instrumentation , and powerful bioinformatics tools . the goals of tissue proteomic research focus on early or more accurate diagnosis , improvement of therapeutic strategies , and better evaluation of prognosis and/or prevention of a given disease , as well as on the identification of novel drug targets based upon the differential protein expression between control and case groups for the disease . a major hurdle in tissue proteomics analysis is the variability observed among the tissue samples due to the heterogeneity of tumor tissues containing cancer cells , as well as inflammatory , vascular , and connective tissue cells . detailed proteomic analyses of these clinically valuable samples require meticulous preparation procedures , which is a major current focus of tissue proteomics . so far , tissue proteomics has uncovered large numbers of proteins with altered expressions in tumor tissues by analyzing both fresh frozen biopsy samples and archived tumor tissues stabilized with formalin fixation and paraffin embedding ( ffpe ) . however , very few , if any , of these proteins have actually become novel diagnostic biomarkers or therapeutic targets . as illustrated in the examples below , most tissue proteomic studies remain as preliminary investigations that identified differentially expressed proteins in diseased tissues . while these proteins could be potential prognostic markers or therapeutic targets , there is a general lack of in - depth functional and validated studies that follow up on these initial screening results . tissue proteomics has been employed quite frequently in the study of lung cancer for molecular mechanism elucidation as well as for novel drug target discovery.121126 peng et al123 studied the protein profile changes between human pulmonary adenocarcinoma tissues and paired surrounding normal tissue with two - dimensional ( 2d ) gel electrophoresis and esi - q - tof ( electrospray ionization , quadrupole , time - of - flight ) ms / ms instruments . thirty - two differentially expressed proteins ( > 2-fold change ; p<0.05 ) were identified in pulmonary adenocarcinoma when compared to normal tissues . knockdown of pyruvate kinase isozyme 2 ( pkm2 ) , one of the overexpressed proteins , led to a significant suppression of cell growth , to the induction of apoptosis in vitro , and to tumor growth inhibition in vivo . moreover , the shrna - expressing plasmid targeting cofilin-1 , another over - expressed protein , significantly inhibited tumor metastases and prolonged survival in vivo . this tissue proteomic analysis and validation indicated that pkm2 and cofilin-1 could be potential therapeutic targets for pulmonary adenocarcinoma . it should be noted that small molecule inhibitors of pkm2 were identified and shown to inhibit cancer cell glycolysis and increase cancer cell death following the loss of growth factor signaling,127 which was consistent with the proteomics - driven conclusion . cofilin-1 has long been associated with increased tumor metastasis due to its role in regulating cytoskeleton dynamics,128 and the inhibition of cofilin-1 by small molecule inhibitors has been demonstrated to enhance actin depolymerization.129 in another example , carretero et al124 performed an integrated genomic and proteomic study for the identification of genes and phosphoprotein status associated with lkb1 loss and progression to invasive and metastatic lung tumors in primary and metastatic de novo lung cancers . phosphoproteomic analysis determined that two key modulators of focal adhesion dynamics , src and focal adhesion kinase , are upregulated by lkb1 loss during nsclc progression . moreover , the combined inhibition of src , phosphoinositide 3-kinase , and mek1/2 resulted in a synergistic tumor regression . these results point towards a mechanism underlying the increased propensity for metastases seen in lkb1-deficient lung tumors , and they identified the src signaling pathway as a molecularly targetable pathway for the treatment of lkb1-deficient nsclc in humans . therefore , development of therapeutic src inhibitors , including dasatinib and saracatinib , will be a valid therapeutic strategy for the treatment of lkb1-deficient nsclc . proteomic analysis of pca tissues associated with multistage tumor progression also provides a valuable source of clinically relevant biomarkers and novel therapeutic targets.130,131 for example , ummanni et al132 recently reported the differential protein expression patterns from histologically characterized pca tumor tissues and surrounding benign tissues of individual pca patients based upon 2d differential gel electrophoresis coupled with ms . the study identified 118 protein spots differentially expressed in cancer ( n=24 ) when compared to benign ( n=21 ) prostate tissues adjacent to cancerous tissues . analysis of these gel spots by matrix - assisted laser desorption / ionization time - of - flight ms / ms revealed 79 unique proteins . moreover , system biology analysis of proteomic results revealed several novel drug targets of pca development and/or progression including eif4a3 , ddah1 , arg2 , prdx3 , and prdx4 , although functional validation of individual targets have yet to be performed . it is evident that proteomic analysis of multistage pca tissues could provide new insights into pca progression and potentially lead to the design of novel diagnostic and therapeutic strategies . efficient peptide / protein extraction approaches are crucial to the success of tissue proteomic analysis . two major strategies of mining proteomic information from ffpe archive tissue samples have been developed.133138 one strategy aimed to recover full - length proteins by heat treatment in suitable buffers with consequent reversion of the formaldehyde - induced cross - links . the operation conditions have been optimized by various labs since shi et al139 initially established this method . application of high temperatures and the addition of detergent sodium dodecyl sulfate were indicated as two critical conditions for enhanced protein extraction yields.140142 because the protein was deposited in large amounts as insoluble , densely packed aggregates , applications of 40,000 psi pressure were reported to recover 96% of proteins from a tissue surrogate model , compared with a 26% recovery rate at 14.7 psi.138,143 another major strategy is based upon the direct proteolytic digestion of intact ffpe archive tissue samples , followed by liquid chromatography ms / ms characterization of the complex peptide mixture.144 tissue solubilization can be achieved in various buffers including sodium dodecyl sulfate dithiothreitol,145 radioimmunoprecipitation assay,146 acetonitrile ammonium bicarbonate,147 tris - hcl148 under different temperatures , and ph conditions for the optimal yield of peptides . as a successful example , hwang et al147 characterized 428 prostate - expressed proteins from ffpe archive tissue samples for the discovery of pca biomarkers and potential drug targets using the shotgun approach . results to date demonstrate that direct trypsin protein digestion is an effective sample preparation strategy for proteomic analysis of ffpe archive tissues . approximately 70% of diagnosed breast cancers express the estrogen receptor ( er+ ) whereas er breast cancers are not well differentiated and clinically tend to be more aggressive.149152 a global proteomic characterization and quantitative comparison of er+ and er breast tumors were recently performed on fresh frozen breast tumor tissues.153 the study identified 2,995 unique proteins including a number of receptor tyrosine kinases and intracellular kinases that are abundantly expressed in both er+ and er breast cancer tissues . using a label - free quantitative approach , 236 proteins were found differentially expressed between er+ and er breast tumors . compared with er breast tumors , 141 proteins were selectively upregulated , while 95 proteins were downregulated in er+ tumors . molecular function analysis of gene ontology showed that the dehydrogenase , reductase , cytoskeletal proteins , extracellular matrix , hydrolase , and lyase categories were significantly enriched in er+ breast tumors , whereas selected calcium - binding proteins , membrane traffic proteins , and cytoskeletal proteins were enriched in er tumors . biological process and pathway analysis indicated that proteins related to amino acid metabolism , proteasome , and fatty acid metabolism were overexpressed in er+ tumors , while proteins related to the glycolysis pathway were overexpressed in er tumors . given the clinical challenges in treating er breast cancer , such in vivo findings of differentially expressed proteins in er tumors are especially significant because of their pathological relevance . these proteins may serve as potential therapeutic targets for er breast cancer if functionally validated . recently , cabezn et al154 used a systematic 2d gel - based proteomic profiling strategy , applied to the analysis of 78 fresh triple - negative breast cancer ( tnbc ) tissue biopsies , in combination with a three - tier orthogonal technology ( 2d polyacrylamide gel electrophoresis / silver staining coupled with ms , 2d western blotting , and immunohistochemistry ) approach , they identified and validated one specific protein , mage - a4 , which was expressed in a significant fraction of tnbc and her2-positive / er lesions . the existence of immunotherapeutic approaches specifically targeting this protein , or mage - a protein family members , provides novel management options for tnbc and her2-positive / er patients bearing mage - a4 positive tumors . although promising results have been reported , there has yet to be a breakthrough that is close to becoming clinically applicable . the challenge will be to perform robustly designed , large retrospective studies including independent validation sets , followed by prospective validation studies to demonstrate the clinical benefits for patients . the development of sirna in vivo techniques will greatly speed up selection of useful proteins . characterization of discriminator proteins will provide new molecularly targeted anticancer drugs . faced with the complexity of cancer tissue proteomics and its great clinical potential , a global coordination of ongoing efforts appears to be crucial . in this review we provided an overview of three major approaches of proteomics employed in discovering potential therapeutic targets for cancer . target identification in resistant cancer populations is one of the most promising tools for drug target identification , and it can uncover multiple drug targets for combinatorial therapeutic solutions to combat resistance and achieve longer - term improvement in disease outcomes . chemical proteomics approaches not only identify protein targets for drugs that exert known biological activities , but they can also discover previously unknown targets for drugs of known moa . the tissue proteomics approach offers the advantage of direct clinical relevance and can complement mechanism - based approaches like chemical proteomics . proteomics continues to evolve rapidly both in instrumental advancement and innovative applications in biomedical research , particularly in the field of cancer . the complexity of cancer biology requires not only in - depth functional studies for mechanistic elucidation , but also unbiased system approaches for a global view of interactive signaling networks that reflect the cancer disease stage . cancer proteomics has uncovered a phenomenal number of cancer - specific proteomic alterations , shedding light on previously unknown mechanisms in tumorigenesis , cancer progression , and metastasis . perhaps more prominently , proteomics has provided an unmatched wealth of potential biomarkers that could be clinically used for the diagnosis and prediction of treatment responses . the utility of proteomics as a valuable tool for the discovery of new cancer treatment targets has been increasingly recognized in recent years . we note the remarkable progress in several directions when cancer proteomics plays an important role in drug discovery , as illustrated in figure 2 . novel proteomics methodologies , both ms - based and non - ms - based , continued to be optimized and perfected using high - resolution and high - speed ms , simplified and robust array technologies for tumor tissue , cell , subcellular compartment , and other relevant biological samples . proteomics has been increasingly used to elucidate the mechanisms of drug resistance in cancer with the hope that novel protein targets could emerge for therapeutic intervention of the resistant disease . in other innovative frontiers , chemical proteomics studies examining drug - induced biological perturbations often reveal new clinically relevant biomarkers and drug targets ; in dissecting mechanisms of cancer progression , proteomics also identified previously unknown or unconsidered druggable targets . however , despite the tremendous progress and potential of proteomics for target discovery , there are serious challenges . a lack of coordinated efforts to follow up with the proteomic discovery of novel therapeutic targets largely accounts for the current gap between drug development ( design , synthesis , and optimization ) and new drug targets emerging from numerous proteomic studies of cancer . most of the proteins identified in the proteomic analyses of drug resistance have not been validated for their roles in specific disease processes and their potential in clinical use . functional studies involving in vitro manipulations of gene expression using specific pharmacological inhibitors , antisense rna , rna interference , or gene knockout experiments should be an integral part of a proteomic study for target discovery . future investigations should place more emphasis on functional studies and on the clinical validation of novel targets for their translation in clinical trials with greater speed and higher success rates . proteomics is also expected to play a major role in the preclinical and clinical research of targeted and combinatorial therapies .
in this work , we introduce a free - standing , vertically aligned conductive polypyrrole ( ppy ) architecture that can serve as a high - capacity drug reservoir . this novel geometric organization of ppy provides a new platform for improving the drug - loading efficiency . most importantly , we present the first formal evidence that an impregnated drug ( dexamethasone , dex ) can be released on demand by a focal , pulsatile electromagnetic field ( emf ) . this remotely controlled , on off switchable polymer system provides a framework for implantable constructs that can be placed in critical areas of the body without any physical contact ( such as percutaneous electrodes ) with the ppy , contributing to a low foreign body footprint . we demonstrate this possibility by using a bv-2 microglia culture model in which reactive oxygen species ( ros ) and inducible nitric oxide synthase ( inos ) expression was attenuated in response to dex released from emf - stimulated ppy .	there are growing clinical demands for controlled and sustained drug release systems to serve as implantable devices for patients with acute and chronic diseases . under these circumstances , it is not surprising that intelligent materials have emerged as a promising strategy for drug delivery . for example , many efforts have been directed toward using various stimuli - responsive biomaterials as on off controllable drug carriers in which the bioactive cargos are released via changes in ph , temperature , or input of electrical or uv energy . at the present time , electrical stimulation appears to be one of the more suitable approaches for clinical translation in that ( 1 ) the electrical signal can be triggered using portable equipment , not requiring significant cost or sophisticated technologies , and ( 2 ) the generated signal can be tuned using a variety of exposure times and current intensities . in this regard , conductive polymers have emerged as one of the more useful drug - delivery platforms . in particular , polypyrrole ( ppy ) has become a candidate material due to its lack of toxicity , favorable biocompatibility , and reversible electrochemical properties . for instance , polypyrrole demonstrated excellent in vivo biocompatibility , with results similar to teflon when implanted as a neural prosthetic . both glial and neuronal cells were found to be in intimate contact with the ppy material . other studies noted that ppy extracts exhibited no hemolytic , allergenic , or mutagenic properties whereas sciatic nerve implants elicited only a minor inflammatory response 6 months postimplantation . functionally , the electrostatic interaction of ppy in response to electric current provides a controllable switch for the release of tethered cargo , providing in situ delivery of nerve growth factors , anti - inflammatory drugs , or adenosine triphosphate . prior investigations demonstrate that time- and site - specific release profiles can be obtained by modifying electrical or magnetic pulse patterns and durations . electromagnetic fields ( emf ) have been further discussed as another potential form of stimulus for drug delivery and was first realized in carbon nanotubes . we have previously outlined the fabrication and physiochemical details that advance the potential of ppy in medical practice . however , two obstacles that prevent the practical use of the ppy polymer systems are the following : ( 1 ) the amount of a drug s cargo is limited when using typical flat thin - film fabrication and ( 2 ) delivery of the cargo within the human body requires percutaneous electrodes to deliver the required level of electric current ( i.e. , a physical electrical contact with the ppy substrate ) . this latter obstacle must be understood in the context of chronic applications where drug release may be desirable over many days until the supply within the film is exhausted . during this time , percutaneous wires carry the possibility of infection by retrograde tracking along the insertion path where normal movement vitiates a perfect seal between tissues and the insulated electrodes . in this work , we detail a new ppy paradigm that overcomes the limits of payload and invasive delivery . we demonstrate that a three - dimensionally nanostructured ppy platform impregnated with a model test drug ( dexamethasone , dex ) exhibits outstanding drug loading efficiency . moreover , noninvasive and on - demand drug release has been demonstrated by exposing the ppy nanowires to high - frequency pulsed electromagnetic fields ( emf ) . subsequent studies using a lipopolysaccharide - challenged bv-2 glial cell line showed that the dex released by emf stimulation remained bioactive and ameliorated both oxidative damage and the inflammatory response . the putative inductive coupling between the dex - doped polypyrrole nanowires ( dex / ppynws ) and emfs bypasses the requirement for direct electrical contact with ppy and opens the door to ppy embodiments that can be placed in vivo in which the cargo can be delivered controllably and noninvasively for many weeks . ppy flat films and nanowires were fabricated using common electropolymerization techniques . the first step in this manufacturing process was the preparation of the templates . for flat ppy films , indium tin oxide ( ito ) glass slides with 515 resistivity ( delta technologies ) were washed in acetone for 30 min , followed by ethanol and milli - q water in an ultrasonic bath . for ppynws experiments , an anodic aluminum oxide template ( aao , figure 1 ) with a 0.2 m pore size and 60 m thickness ( whatman ) was obtained . the aao templates were subsequently coated with a 100-nm - thick gold layer on one side using a varian e - beam evaporator . briefly , an aqueous mixture consisting of 0.2 m pyrrole ( py , sigma ) , 0.025 m dexamethasone 21-phosphate disodium salt ( dex , sigma ) , and 0.05 m 10 nm nanoxact spherical gold nanoparticles ( aunps , nanocomposix , ca ) was mixed . for comparison , 0.2 m pyrrole , 0.025 m dex , and 0.1 m poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate ) ( pss , sigma ) were also mixed for synthesis . note that for the in vitro experiment the concentration of dex within the solutions was decreased to 5 mm . prepared aao templates were subsequently incubated into their respective mixtures for 30 min . cleaned ito slides and aao templates were separately connected to the working electrode from a ch instruments model 604 electrochemical analyzer / workstation , with a platinum counter electrode and ag / agcl reference electrode placed in the synthesis solution . the one - step electropolymerization of dex / ppy was accomplished by applying a constant potential of 1 v using a potentiostat . following polymerization , the final films were rinsed thoroughly with milli - q water for 5 min . the aao templates were removed by placing the films in 3 m sodium hydroxide and then rinsed with milli - q water . each cutaway diagram is an illustration of the aao template . at the far left the third step illustrates the filling and diffusion of the solution consisting of pyrrole monomer , aunps or pss , and dex in the template pores . the fourth step is the dex - conjugated polypyrrole electropolymerization process . finally ( far right ) , the ppy nws are shown attached to the gold base after the dissolution of the aao template with sodium hydroxide . ppy samples were prepared by first sputter coating with platinum for 60 s. sem images were taken with an fei nova nano sem via field emission scanning using et and tld at high vacuum at an acceleration voltage of 5 kv . tem images were taken using a fei / philips cm-10 transmission electron microscope operated at 100 kv , a 200 m condenser aperture , and a 70 m objective aperture . multiple factors must be considered when designing the emf stimulation system and pulse conditions . these parameters include the waveform shape , pulse duration , pulse magnitude , duty cycle , and so forth . we originally chose a narrow pulse width ( 500 ns ) stimulating regime as it allowed us to experiment with various coil geometries with minimal power consumption . the square wave was chosen in order to maximize the induced electric fields within the ppy since that is the hypothesized stimulus for drug release . our original pilot measurements were made using a three - turn coil which had a very low inductance allowing testing that set the limits for overheating . this data ( not shown ) then permitted the construction of a 15-turn coil , which was used in all experiments reported here . briefly , awg 16 copper wire was wound 15 times to create a 15-turn coil . the geometry of the coil was 2.3 2.8 cm inside dimensions and 3.0 3.8 cm outside dimensions . the coil was then stimulated with a pulsing regime using a custom - made circuit . each input pulse was on only long enough to saturate the coil and then was turned off for the duration required to unload the coil completely ( 10 s ) . additionally , we tested several stimulation patterns such as oscillating the polarity , grouping the same polarity pulses in different temporal patterns , and so forth while maintaining a 5% duty cycle . as we did not find significant differences in drug release in the initial trials ( data not shown ) , we utilized the waveform described in figure s2 in order to minimize heat generation . ( a ) the top illustration shows the integrated system used to create the electromagnetic field ( emf ) to induce ppynws activity . the stimulator is connected to a wire - wound coil in which a standard cuvette is placed in the center . following emf stimulation , aliquots of the solution were assayed for drug concentration via uv vis spectrophotometry . the left inset describes the dimensions of the stimulating coil , where h = 1.2 cm , l = 3.8 cm , and w = 3.0 cm . the recordings in ( b ) , left and right , are probe measurements of the magnetic fields ( mf ) and electric fields ( ef ) at the center of the coil ( 36 g and 4000 v / m , respectively ) . the insets describe the idealized mf and ef waveforms based on the input square - pulse stimulation regime . using the waveform patterns in figure s1 as the input signal , the real time magnetic and electric field outputs from the stimulation coil were measured using high - frequency emc 100b ( magnetic ) and 100d ( electric ) probes ( beehive electronics , ca ) . probes were connected to a tektronics tds 2012b oscilloscope and terminated with a 50 resistor . for data collection , each probe was positioned in the stimulation coil center ( origin ) . the probes were directional and oriented in the direction of the maximum expected reading . this value was taken to be the output probe value and was converted to dbm . the recorded data points from the oscilloscope were imported into ms excel in which the fast fourier transform ( fft ) of the waveforms was calculated . from the ffts , the fundamental frequency and with the output dbm power value were used with the antenna gain equation ( figure s1 ) provided for each probe to estimate ac field magnitudes . both potentiostat electrical stimulation and the setup was the same as for the electropolymerization process with three electrodes , but the dex - conjugated ppy film was connected to a working electrode and subsequently placed in pbs solution containing 0.80% w / v nacl . a constant voltage of 0.1 v was applied for 5 h using a ch instruments model 604 electrochemical analyzer / workstation . for comparison , the dex / ppy platforms were also stimulated by an induced emf generated by our custom circuit and coil stimulator system . the supernatants were collected after 1 , 3 , and 5 h of stimulation for both the potentiostat and the pulsed magnetic field generator . figure 2 depicts the experimental setup and the essential components of the emf stimulation system used to remotely trigger ppy drug release . the ppy samples were placed in a 12.5 mm 12.5 mm 45 mm standard cuvette filled with phosphate buffer solution . the cuvette was positioned in the center void of the stimulation coil but was not in physical contact with the coil . the coil was subsequently energized using square wave pulse trains as described in figure s1 . experiments comparing the dex - conjugated polypyrrole compositions and the drug release effects of stimulation were continued for up to 16 days using emf stimulation . to test if the stimulated release of dex might be influenced by the heating of stimulated samples , we placed the dex - doped flat polypyrrole templates within a controlled incubator at 37 c . this temperature was slightly higher than ambient temperature in the vicinity of the active coil , which was measured to be about 30 c . for all 16 days of dex release , the sample solutions were collected after 1 , 3 , 5 , 7 , 10 , 13 , and 16 days of continuous emf stimulation ( n = 5 ) . a calibration curve of dex ( y(od ) = 23.8x 0.04 ) was also prepared to detect the drug release . to determine the characteristics of pulsatile emf stimulation , on and off experiments were performed on polypyrrole nanowires synthesized with gold nanoparticle ( ppynws aunps ) samples . the dex release profile recordings were begun after 10 h of stimulation by the pulsed emf . then , pulsed emf stimulation was turned on ( referred to as the on time ) for 2 h and discontinued ( off time ) for 2 h. this regimen was continued for four cycles for each sample . ppy samples of about 0.51.0 cm were prepared and submitted for xps analysis . the surface chemical composition of the samples was analyzed by x - ray photoelectron spectroscopy ( xps ) using a kratos axix ultra dld spectrometer . the survey and high - resolution spectra were obtained in constant pass energy mode with pass energies of 160 and 20 ev , respectively ( survey , 1 ev / step ; high - resolution spectra , 0.05 ev / step ) . the bv-2 cells were cultured in 75 cm flasks in dulbecco s modified eagle s medium ( sigma - aldrich ) with 10% fetal bovine serum , 100 iu / ml penicillin , and 100 g / ml streptomycin . cells were maintained at 37 c in a humidified incubator with 5% co2 , harvested by trypsinizing ( 0.25% trysin / edta in pbs ) , and reseeded ( at approximately 1 10 density ) in 12-well dishes for 24 h to detect ros . cells were incubated in the medium containing 1g / ml lipopolysaccharides ( lps ) from escherichia coli 026:b6 ( sigma - aldrich ) for 6 h. then , bv-2 cells were treated with 1 g / ml dex after the application of lps for 1 h. to detect dex release by pulsed emf , ppynws - aunps films ( approximately 30 - 40 mm in unit area ) were stimulated for 30 min and 1 h after lps treatment in 1 h increments . in these experiments , the coil was placed flat directly below the petri dish , aligned with the well and touching the bottom surface of the dish . after 6 h of lps treatment , cells were trypsinized , centrifuged , and resuspended in a 10 m oxidative stress indicator ( cm - h2dcfda , molecular probes ) in pbs . suspended cells were then placed in the incubator for 1 h , and the measurement of ros was performed with a plate reader at 480 nm/530 nm . measurement was also performed with a fluorescence microscope ( olympus ix 81 inverted ) . the cells were illuminated with an x - cite series 120pcq fluorescence illumination source . additionally , another strong oxidizing reagent , hydrogen peroxide ( h2o2 ) , was used to confirm the lps results ( oxiselect intracellular ros assay kit ; cell biolabs , inc . ) . briefly , bv-2 cells were seeded in black 96-well plates for 12 h and then incubated in 1 mm dcfh - da solution for 1 h. hydrogen peroxide ( 20 m ) was incubated in these plates for 30 min , and then dex treatments were applied for 15 min . for emf - stimulated dex release , the ppynws - aunps films were divided into smaller pieces ( approximately 30 mm ) , placed in each well , and stimulated for 15 min . in these instances , the coil was placed below the 96-well plate touching the bottom surface , with wells in a 3 3 matrix being stimulated . bv-2 cells were cultured on poly - d - lysine ( sigma ) precoated round cover glasses ( no . 1.5 thickness , 12 mm ) in 12-well plates for 24 h. after experimental treatment with lps , dex , and released dex , bv-2 cells were fixed with 4% paraformaldehyde for 30 min at room temperature . a blocking solution , 1% albumin from bovine serum ( sigma - aldrich ) , was applied to the samples for 1 h. antinitric oxide synthase ii antibody ( emd millipore ) was diluted in a ratio of 1:400 with antibody dilution buffer ( sigma ) . cells were then incubated in a diluted primary antibody solution overnight at 4 c . the next day , these cells were incubated in 1:100 diluted cy3 conjugated goat antirabbit igg antibody solution ( emd millipore ) for 2 h. dried cover glasses were mounted on glass slides using a vectashield hardset mounting medium with dapi ( vector lab ) . these samples were all imaged with a confocal microscope ( leica sp5/sted / mp system ) . all of the data are shown with the standard error of the mean sem . comparative tests used were the conventional student s t test and one - way anova , with significance determined by a p value of 0.05 . nanowire architecture was revealed by sem and tem ( figures 3 and 4 ) . polypyrrole wires ( 10 m long ) were completed in about 13001400 s of deposition using the potentiostat ( figure 2a ) , while shorter lengths were realized for shorter deposition times ( figure s3b ) . the desired structure of ppynws was achieved for 200300 s of deposition ( figure 3b ) . as an aside , smaller ppynws ppynws ( 200 nm long ) were fabricated using different aao templates with 0.02 m pores ( figure s3a ) , but these were not used in this study . the free - standing , vertically aligned ppynws were generally arranged into mats in unit areas of up to 1 to 2 cm . ( a ) scanning electron micrograph showing a top ( dorsal ) view of polypyrrole wires ( 200 nm diameter and 10 m length ) produced with 13001400 s of deposition . ( b ) a similar micrograph where 200300 s of deposition was used . scale bars : ( a ) 10 m and ( b ) 2 m . figure 4a , b shows the distribution of gold nanoparticles within the bulk of 500-nm- to 2-m - long ppynws . note that several gold bases are shown due to the folding of the ppynws . ( b ) higher - magnification tem of individual nanowires showing the details of gold nanoparticle deposition . these wires range from approximately 500 to 2000 nm in length and 150 nm in diameter . the real - time magnetic and electric field output waveforms at the coil center were plotted in figure 2 . the measurements revealed that the magnetic field output was similar to the input field , with some oscillation noise present in the square waveform . fft decomposition of the measured signal showed that the fundamental frequency of the magnetic field was 3.2 mhz . this converts to an average peak amplitude of 36 g when using the antenna gain equation supplied by the probe vendor ( figure s1 ) . moreover , the amplitude of the magnetic field did not vary by more than 20% within the coil . the highest field amplitudes were located near the corners in the x y plane containing the coil center . along the z axis , there was a small decrease in intensity as the probe was moved away from the coil center . overall , the magnetic field was focused and concentrated within the internal space of the coil . however , outside of the coil , the magnetic field became divergent and weak , with the field roughly following a cubic decay . therefore , ppy drug - release experiments were tested with the films situated within the coil . on the basis of the obtained mf probe readings , the ppy films were exposed to peak magnetic fields within the range of 2540 g. similarly , the raw electric field data obtained for each coil is also depicted in figure 2b . note that the measured electric field corresponded to the derivative of the measured magnetic field , as expected by faraday s law . the electric field waveform exhibited oscillatory behavior with sharp peaks primarily concentrated in the ramp - up and switch off phases of the emf . the 15-turn coil produced a peak e field of 4700 v / m and a fundamental frequency of 65 mhz . thus , the estimated electric field magnitude in which the ppy fabrications resided is in the 30005000 v / m range for the 15-turn coils . we emphasize that due to the high - frequency pulsed nature of the stimulation pattern these are the estimated peak emf values during the 500 ns pulse duration . the time - averaged emf values are much lower when considering the duty cycle and would be less than 5% of these values . the distance dependence of the electric field from the coil center followed that of the magnetic field . note that for bv-2 cell culture experiments , the coil was placed on the bottom of the petri dish since it was not possible to place the culture chamber within the coil . in such situations , the measured peak fields to which the ppy substrates were exposed were between 320 g and 3002500 v / m for the mf and ef , respectively . drug release characteristics of the ppynws to conventional flat ppy films are shown in figure 5 . ( a ) the release profiles of dex from both a conventional flat ppy film and ppy nws within 5 h , with one group stimulated electrically using a potentiostat and the others by emf . again , there was only marginal release of dex ( < 0.5 mg / cm ) from flat ppy films . ( c ) active ( emf stimulation ) vs passive ( no stimulation ) of both flat ppy films and ppynws . note that an extraordinary release of dex occurs after emf stimulation of the ppynw - aunps . the small amount of dex detected at the off times was likely due to passive diffusion from the source . * p 0.05 , * * p 0.01 , and * * * p 0.001 . figure 5a reveals the results of drug release monitored over a 5 h period . potentiostat stimulation of flat ppy - aunps was not significantly different from that of emf stimulation . upon exposure to emf stimulation , there was a statistically significant increase in the release levels of ppynws compared to that of the flat ppy film ( * p 0.05 ) . also note that the emf stimulation of ppynws - aunps resulted in more release of dex than did potentiostat stimulation . the inclusion of gold nanoparticles greatly improved the loading capacity and the rate of release . this trend was preserved in both flat ppy thin films and in the ppynws , when compared to the pss - doped ppy films . at 1 , 7 , and 16 days , these differences were almost double for both ppy platforms ( * p 0.05 between ppynws and * * p 0.01 between flat ppy ) . also , ppynws showed a very significant enhancement of dex release triggered by emf stimulation when compared to flat ppy ( on days 1 and 16 , * * p 0.01 , and on day 7 , * * * p 0.001 between aunps ; at days 7 and 16 , * * p 0.01 between psss studied , figure 5b ) . background release studies ( no stimulation ) showed that the outward diffusion of dex was below 0.11 mg / cm for ppynws and 0.3 mg / cm for flat ppy films on the last day of recording . this reveals a significant drug release of emf stimulation compared to that without emf stimulation . ( on day 1 , * * p 0.01 , and on days 7 and 16 , * * * p 0.001 between ppynws ; on day 1 , * p 0.05 , and on days 7 and 16 , * * p 0.01 between flat ppy films , figure 5c ) . figure s4 depicts the dex release profiles of flat ppy as a function of temperature . some passive background release was found , but it did not reach the levels of active emf stimulation . further switching experiments where the emf was turned on for 2 h and then turned off for 2 h are shown in figure 5d . in such cases , dex release fell off precipitously after the removal of emf stimulation and was regained when emf coupling was resumed . such consistency over multiple unloading cycles demonstrated excellent reproducibility and reversibility that is a hallmark of controlled release . x - ray photoelectron spectroscopy ( xps ) was used to verify the existence of dex and to verify the escape of entrapped drug ( figure 6 ) . xps surface analysis was conducted on the ppynws before and after ( 16 days ) pulsed electromagnetic stimulation . as expected , high - resolution spectra of f 1s and p 2p revealed changes in the peak magnitudes , denoting an obvious release of dex upon exposure to the emf . specifically , these spectrographs detail signatures of fluorine and phosphorus elements in the dex molecule , which essentially vanish after emf stimulation . for ppynws - aunps , the prestimulation f 1s and p 2p atomic percentages were 1.53 and 1.54% , respectively , while the poststimulations results were 0.00 and 0.17% . for ppynws - pss , the prestimulation f 1s and p 2p atomic percentages were 0.60 and 1.31% , respectively , while the poststimulations results were 0.00 and 0.15% xps spectra for the surface analysis of the dex presence on ppy templates before and after pulsed emf stimulation . ( a ) xps recording of dex - doped ppynws - aunps before ( green ) and after ( red ) dex release by emf . ( b ) xps data of dex - doped ppynws - pss before ( green ) and after ( red ) 16 days of emf stimulation . these show details of signature fluorine and phosphorus elements in the dex molecule before stimulation , respectively . note that these elemental signatures vanish after emf stimulation , indicative of dex release from the surface . the bottom graph shows the molecular structure of dex , with fluorine ( f ) and phosphorus ( p ) highlighted in red . two groups of toxin - challenged murine neonatal microglial cells ( bv-2 ) were used to evaluate ros production and to determine whether such damaged cells could be rescued via emf - associated drug release . in one group , the challenge was bacterially derived lps ( figure 7a g ) , and in the other , the cells were directly insulted with hydrogen peroxide ( figure 7h ) . ( b ) bv-2 cells insulted by lps , with intense green labeling of ros by 2,7-dichlorodihydrofluorescein ( dcf ) . ( c ) ros was eliminated by the direct introduction of 1 g / ml dex suspended in the medium . ( d ) 30 min of emf stimulation from dex - doped ppynws - aunps , demonstrating reduced ros production . ( e ) with 1 h of emf stimulation , ros production was nearly undetectable . ( f ) in contrast , ppynws not subjected to emf stimulation again showed significant ros production in the tested cell population . ( g ) corresponding graph describing the quantitation of ros production in panels a f . ( h ) additional ros data using h2o2 as a positive control to induce ros . the graph confirms that both applied and stimulated dex suppressed ros production and that dex retained its bioactivity after emf stimulation . * p 0.05 and * * p 0.01 . bv-2 cells treated with lps released pro - inflammatory cytokines and ros via mapk signaling pathways . cm - h2dcfda is one such indicator compound for ros and was used as a metric for ros production . lps ( 1 g / ml)-treated cells exhibited bright green fluorescence in the cytoplasm , marking the production of significant oxidative stress ( figure 7b ) . the addition of dex ( 1 g / ml ) to these lps - induced microglia effectively suppressed ros production during the inflammatory cascade ( figure 7c ) . the dex - doped ppynws - aunps platform also suppressed inflammation byproducts during emf stimulation ( figure 7e ) . a weaker inhibition of ros activity occurred when shorter stimulation times were applied ( figure 7d ) . in contrast , nonstimulated dex / ppynws - aunps films did not show signs of ros scavenging ( figure 7f , g ) . similarly , exposure to 20 g / ml h2o2 resulted in more ros byproducts in bv-2 cells ( figure 7h ) . this enhanced production of ros was reduced either by direct application of 10 g / ml dex or by 15 min of emf stimulation to the dex - coupled ppynws - aunps . microglial cells were stained intensely with cy3 ( red fluorescent signal ) when exposed to lps in a dose - dependent manner ( figure 8) . inos expression in bv-2 cells after lps challenge ( without stimulation ) also showed positive cy3 staining , which is in agreement with the ros results ( figure 7 ) . in contrast , emf stimulation of dex - conjugated ppynws - aunps resulted in a strong suppression of inos , indicating a reduced level of nitric oxide . inos expression in bv-2 cells is shown with red fluorescence ( cy3 column ) . the row labeled as control does not show any inos expression due to the absence of lps treatment . in contrast , the row lps 10g / ml shows significant upregulation of inos , and the merged confocal micrograph details that the staining is primarily localized in the cytoplasm . / ml shows less upregulation at a reduced lps concentration while ppynws - aunps not stimulated with emf ( dex w / o sti ) showed a similar response . / ml / dex 1g / ml ) , results were comparable to the untreated control and highlighted the suppression of inos to lps - challenged bv-2 cells . finally , ppynws - aunps stimulated with emf ( 1 h ) produced inos results comparable to direct dex application . the electroactive properties and biocompatibility of ppy are desirable features in designing programmable drug - delivery systems . previously , our group used topographically modified ppy films that increased the available surface area for drug inclusion and release . while the drug capacity was improved , these films were difficult to fabricate and highly porous , making them susceptible to damage . we have thus introduced another form of conductive ppy by shaping the polymer into solid nanowire arrays for further functionalization . moreover , we propose the use of electromagnetic fields as the stimulus for ppy drug release . this is a significant departure from all previous forms of drug - doped ppy , in which direct electrical connection ( i.e. , wire electrodes ) to the ppy was required to induce current flow and subsequent drug release . experimental results showed that the ppynws - aunps possessed excellent drug - carrying capabilities as well as a controllable switching response to electromagnetic fields . xps analysis confirmed that the model drug , dexamethasone , was successfully incorporated onto the surface of the ppy nanowires . when exposed to pulsatile emfs , the dex cargo was released from the ppy surface , a process that was verified by changes in the surface chemistry and the free drug concentration . the amount of drug that could be eluted from emf stimulation was superior to direct stimulation via a potentiostat . while the mechanism of emf - coupled drug release is still unclear , we hypothesize that the phenomenon is similar to ppy stimulation via direct current . for instance , it is well known that ppy undergoes an electrical and conformational change via an oxidation / reduction reaction in response to applied voltages . furthermore , the electroactive ppy swells during oxidation and shrinks ( mechanical actuation ) during reduction to satisfy charge balance . this reversible volume change along with the electrostatics of moving charges within the ppy governs how charged molecules move in and out of the ppy ( i.e. , drug deposition and elution ) . a multitude of cytokines / drugs such as nerve growth factors , analgesics , and adenosine triphosphate have been released in situ based on this functionality . in the proposed polymer platform , charge balance is obtained when the anionic dex is electrostatically entrapped within the cationic pyrrole chain during the electropolymerization process . when a sample is exposed to a time - varying em field , an oscillating electric current is induced in the ppy and is posited to drive the redox reaction . during the reduction state , charge neutralization of the pyrrole backbone eliminates the electrostatic bonding to the dex , causing the dex to migrate out of the ppy matrix . indeed , prior evidence has shown that dex desorbs from ppy in the reduction state . therefore , the cumulative reduction states that occur during the inductive cycling of ppy facilitate dex movement . the concomitant mechanical actuation or pumping action from the redox reaction may further play a role in forcing the dex out of the ppy . not surprisingly , this polymer electroactive effect should be more pronounced if the conductivity of the ppy is enhanced , which was indeed observed when the ppy nanowires were impregnated with gold . as a result , it appears that dex elution occurs regardless of whether current flow is direct or electromagnetically induced . it has been noted that ppy is susceptible to overoxidation whereby the electroactivity can diminish due to high applied voltages or continued stimulation . this fatigue behavior was not detected as evidenced by the prolonged dex release over a period of 16 days in vitro . the extended electrochemical stability of ppynws - aunps may be partially due to the low duty cycle of the applied em fields and its rapid oscillatory nature or by the enhanced conductivity conferred by the gold nanoparticles . for instance , the addition of multiwalled carbon nanotubes improves the conductivity and electrostability of ppy neural prosthetic coatings . however , induction may cause joule heating and subsequent drug release , an observation that was described for carbon nanotubes . to address passive thermal effects , we performed additional studies at elevated temperature ( 37 c ) . results show that temperature played only a small role in the release kinetics . follow - up cyclical stimulation also revealed that dex was released only during on stimulation states . it is further unlikely that ph changes were responsible for drug elution since these experiments were conducted in physiologically buffered solutions . to verify the bioactivity of the released dex cargo , we performed several cell culture assays using the bv-2 glial line exposed to lipopolysaccharide ( lps ) toxin . lipopolysaccharide - challenged microglia have been shown to induce neurotoxicity through the production of pro - inflammatory mediators such as tumor necrosis factor- , interleukin-1(il-1 ) , ros , and inos , which catalyze the production of nitric oxide ( no ) . since a focus area within our laboratory is neurodegenerative diseases of the central nervous system ( cns ) ( reviewed in ref ( 20 ) ) , we chose this in vitro neuroinflammatory model to assess the therapeutic activity of emf - released dex . the results showed that the addition of dex ( 1 g / ml ) to lps - exposed microglia suppressed ros production . similarly , the dex - doped ppynws - aunps platform also suppressed inflammation byproducts during emf stimulation . in contrast , the nonstimulated dex / ppynws - aunps films did not confer signs of ros scavenging . similarly , exposure to the h2o2 positive control resulted in ros byproducts in bv-2 cells . this production of ros was also reduced either by direct application of 10 g / ml dex or with 15 min of emf stimulation to the dex - coupled ppynws - aunps . these findings were consistent with inos measurements , with a marked reduction in inos expression in the presence of soluble dex . while we can not eliminate the possibility that emf alone may reduce ros - associated damage in bv-2 cells , this is highly unlikely since the nanosecond pulse widths and stimulation times used were much shorter than the emf waveforms commonly employed in bioelectromagnetics . therefore , we conclude that the mitigation of the inflammatory cascade was due to the availability of free dex . even as the proposed geometric scheme of ppy improves with drug - loading capacity , the amount of drug delivered may still be considered to be miniscule in comparison to systemic delivery . rather , the intent of nanoscaled reservoirs is to deliver extraordinarily high concentrations of potent drugs to a localized microenvironment in order to produce therapeutic responses in adjacent , contiguous cells . in essence , nanoconstruction aims to release drugs into only specified target areas while escaping systemic circulation and side effects , a critical advancement in therapies involving potent or toxic drug cargos . examples of such indications include early detected tumors , a restricted lesion in the nervous system ( such as a localized region of acute cns embolism or trauma ) , and the local region of small - scale crush injury to cns or pns axons that completely inhibit action potential propagation across the injury zone . these minor spatial defects produce profound and catastrophic behavioral , functional , and cognitive consequences . here we show that the release of drugs from ppynws can occur for more than 2 weeks and may serve as an ideal candidate for such applications . we finally note that the reported emf stimulation protocol is not fully optimized , and we are refining methods in which this release system can be employed for broad biomedical and pharmaceutical applications . as the putative mode of drug release is emf - ppy induction coupling , we aim to develop oscillating waveforms that maximize the induction current without significant joule heating . the phenomenon of joule heating requires special attention in vivo since the thermal energy must be dissipated . additional design parameters will also account for the attenuation of the em signal through skin and connective tissues . various theoretical and empirical models that consider tissue permeability , conductivity , and imposed emf frequency have been developed and will be used as a reference for design . however , the pulsing frequencies used in the present system are effective in vitro within a couple of centimeters of the coil center and will serve as a basis for further in vivo study . future improvements may also include synthesizing degradable forms of ppy which may be resorbed within the body . nonetheless , we reveal for the first time that a ppy system can be used as a type of programmable drug - delivery reservoir that responds to electromagnetic fields . this action at a distance potentially provides a new direction for noninvasive controlled drug release . conductive polymer polypyrrole was fashioned into a nanowire architecture and doped with the drug dexamethasone . this geometry enabled high - capacity drug entrapment and subsequent sustained release for over 2 weeks . more importantly , the release of dexamethasone could be triggered on demand via an externally applied pulsed electromagnetic field . dexamethsone remained bioactive , as demonstrated by its ability to ameliorate damage to toxin - challenged glial cells . this is the first demonstration of a noninvasive mode of drug delivery using polypyrrole .
background and objectivesthe purpose of the present study was to provide the hearing threshold levels in the elderly korean population , and to compare korean data with that in the international organization for standardization ( iso ) 7029 ( 2000).subjects and methodsdata were collected from a total of 526 ears from 112 males and 151 females aged 60 - 84 years . all participants were screened otologically by the procedure given in iso 8253 - 1 ( 2010).resultsresults showed that the pure - tone average was gradually elevated with increasing age . the amount of hearing loss was greater in males than in females , and the high frequency hearing thresholds were worse than the low frequency hearing thresholds in males and females . the hearing threshold levels were higher at low frequencies in males and at all frequencies in females than the norms of iso 7029 ( 2000).conclusionsresults from this study will be partly used for standardization of hearing thresholds as a function of age in korea and for updating the iso 7029 .	it is well known that hearing levels progressively deteriorate with age which is the most common factor associated with acquired hearing loss . age - related hearing loss is prevalent in the elderly regardless of age , gender , or race . the prevalence of hearing loss is more than 50% in people older than 50 years in the u.s . . in europe , more than 55% of males have a hearing loss of more than 30 db hl by the age of 80 years . age - related hearing loss is characterized that high - frequency sounds are more affected than low - frequency sounds . the deterioration of hearing thresholds is mainly due to aging of the peripheral and central auditory system and exposure to environmental noise . the degree of hearing loss varies among individuals , although the degree of hearing loss is larger in males than in females . the age - related variation in hearing thresholds was defined by international organization for standardization ( iso ) 7029 , providing the expected hearing threshold distribution in population aged from 18 to 70 years . however , iso 7029 was based upon the hearing thresholds for european and north american populations between the 1950s and the 1970s . some authors have indicated the problems with iso 7029 in terms of specific population data , subject selection criteria , outdated calibration and test procedures , and restricted test frequencies . authors have proposed to revise the norms of hearing thresholds as a function of age , and as a result , iso 7029 is currently under revision by the iso technical committee 43 . in that study , 2492 adults ( 1250 males and 1242 females ) with an age range of 20 - 59 years who were otologically normal were included . the primary goal of this study is to provide the distribution of hearing thresholds as a function of age in korean individuals from 60 to 84 years of age who have been screened by the procedure described in the iso 8253 - 1 . the hearing threshold levels in this study were compared with those provided by the iso 7029 . also , the hearing thresholds for each signal frequency according to gender and age groups were compared . we anticipate that these data will be used for preparing new standards of hearing thresholds as a function of age in korea and for updating the iso 7029 under revision . data were collected from a total of 526 ears ( from 112 males and 151 females aged 60 - 84 years ) . the age criteria were stratified into 60 - 64 , 65 - 69 , 70 - 74 , 75 - 79 , and 80 - 84 groups in 5-year intervals as shown in table 1 . all subjects were recruited from various areas in korea ( seoul , chuncheon , gwangju , daegu , and mokpo ) in order to ensure sampling from the entire country . all of the participants received an a - type tympanogram and did not report a history of hearing loss , ear surgery , otologic disease , or occupational noise exposure ( iso 8253 - 1 , 2000 ) . a gsi 61 audiometer ( grason - stadler , eden prairie , mn , usa ) was used for measuring pure - tone thresholds in a soundproof room . according to the criteria of iso 8253 - 1 , the maximum permissible sound pressure level for ambient noise in the room was sufficiently low . the test tones were calibrated first by the method of iso 389 - 5 and 389 - 8 and then presented to participants through tdh-50 headphones ( telephonics , farmingdale , ny , usa ) . a sound level meter ( type 2250-l ; b&k , denmark ) was used for the calibrations with a microphone ( type 4192 ; b&k ) and a preamplifier ( type 2690-a-0s1 ; b&k ) . informed consent was obtained from the participants and a questionnaire was additionally completed before audiometric tests . both ears were tested , and the first ear ( right or left ) was chosen randomly in all subjects . if hearing thresholds in right and left ears differed by more than 20 db hl at a given frequency , only better hearing thresholds in the ear were obtained . pure - tone air conduction audiometry was conducted over the range of 250 - 8000 hz in the order of 1000 , 2000 , 3000 , 4000 , 6000 , 8000 , 500 , and 250 hz with a combined ascending and descending approach following a standardized protocol ( iso 8253 - 1 ) . an audiologist presented stimuli , and the participants were asked to press a button when they heard any beep sound . , armonk , ny , usa ) . the hearing thresholds for each signal frequency for each age band were compared by gender using an independent t - test . the dependent variable was the hearing thresholds for the frequencies , and the independent variable was gender . to compare average hearing thresholds in each age band for males and females , one - way analysis of variance ( anova ) the between factor was each age band , and the independent variable was average hearing threshold . iso 7029 presents the equation hmd , y= ( y-18 years ) for computing the expected age - related hearing loss . h refers to the deviation from the median hearing threshold level at an age of y years based on a reference age of 18 years ( 0 db hl ) . data were collected from a total of 526 ears ( from 112 males and 151 females aged 60 - 84 years ) . the age criteria were stratified into 60 - 64 , 65 - 69 , 70 - 74 , 75 - 79 , and 80 - 84 groups in 5-year intervals as shown in table 1 . all subjects were recruited from various areas in korea ( seoul , chuncheon , gwangju , daegu , and mokpo ) in order to ensure sampling from the entire country . all of the participants received an a - type tympanogram and did not report a history of hearing loss , ear surgery , otologic disease , or occupational noise exposure ( iso 8253 - 1 , 2000 ) . audiometric tests were conducted by trained audiologists . a gsi 61 audiometer ( grason - stadler , eden prairie , mn , usa ) was used for measuring pure - tone thresholds in a soundproof room . according to the criteria of iso 8253 - 1 , the maximum permissible sound pressure level for ambient noise in the room was sufficiently low . the test tones were calibrated first by the method of iso 389 - 5 and 389 - 8 and then presented to participants through tdh-50 headphones ( telephonics , farmingdale , ny , usa ) . a sound level meter ( type 2250-l ; b&k , denmark ) was used for the calibrations with a microphone ( type 4192 ; b&k ) and a preamplifier ( type 2690-a-0s1 ; b&k ) . informed consent was obtained from the participants and a questionnaire was additionally completed before audiometric tests . both ears were tested , and the first ear ( right or left ) was chosen randomly in all subjects . if hearing thresholds in right and left ears differed by more than 20 db hl at a given frequency , only better hearing thresholds in the ear were obtained . pure - tone air conduction audiometry was conducted over the range of 250 - 8000 hz in the order of 1000 , 2000 , 3000 , 4000 , 6000 , 8000 , 500 , and 250 hz with a combined ascending and descending approach following a standardized protocol ( iso 8253 - 1 ) . an audiologist presented stimuli , and the participants were asked to press a button when they heard any beep sound . the hearing thresholds for each signal frequency for each age band were compared by gender using an independent t - test . the dependent variable was the hearing thresholds for the frequencies , and the independent variable was gender . to compare average hearing thresholds in each age band for males and females , one - way analysis of variance ( anova ) the between factor was each age band , and the independent variable was average hearing threshold . iso 7029 presents the equation hmd , y= ( y-18 years ) for computing the expected age - related hearing loss . h refers to the deviation from the median hearing threshold level at an age of y years based on a reference age of 18 years ( 0 db hl ) . table 2 lists the numerical data related to the hearing threshold distributions ( 5 , 10 , 25 , median , 75 , 90 , and 95 percentiles ) for males and females in each age band . the results of the independent t - test showed that there were no significant differences between genders in low frequencies ( 250 and 500 hz ) , but there were significant differences in high signal frequencies . the average hearing thresholds were computed by averaging the hearing sensitivity at 500 , 1000 , and 2000 hz ( table 4 ) , and 4000 , 6000 , and 8000 hz ( table 5 ) . for comparing the mean hearing thresholds of each age band , the independent variable was the average hearing thresholds and the between factor was the age band . results of mean thresholds of 500 , 1000 , and 2000 hz revealed that there were significant differences among the age bands for males [ f(4 , 213)=12.042 , p<0.001 ] and for females [ f(4 , 293)=15.760 , p<0.001 ] . results of mean thresholds of 4000 , 6000 , and 8000 hz also showed that there were significant differences among the age bands for males [ f(4 , 213)=12.478 , p=0.000 ] and for females [ f(4 , 293)=15.760 , p=0.000 ] . table 6 and table 7 provide summary of post hoc tests for mean thresholds of 500 , 1000 , 2000 hz and those of 4000 , 6000 , 8000 hz , respectively . table 8 presents the mean alpha coefficient values for the present study and those for iso 7029 at each signal frequency for males and females . figs . 1 and 2 show the mean hearing thresholds of each age band for the males and females . since iso 7029 does not provide hearing thresholds according to age bands , the median hearing thresholds at each age were computed by the equation and means of the hearing thresholds for each age band were calculated for comparison . results revealed that the elderly korean population had worse hearing thresholds than the estimated hearing thresholds based on the iso 7029 equation applied to the same age band . in particular , korean males had similar hearing thresholds at high frequencies and worse hearing thresholds at low frequencies than those based on the iso 7029 . korean females had worse hearing thresholds at all frequencies than those based on the iso 7029 . table 2 lists the numerical data related to the hearing threshold distributions ( 5 , 10 , 25 , median , 75 , 90 , and 95 percentiles ) for males and females in each age band . the results of the independent t - test showed that there were no significant differences between genders in low frequencies ( 250 and 500 hz ) , but there were significant differences in high signal frequencies . the average hearing thresholds were computed by averaging the hearing sensitivity at 500 , 1000 , and 2000 hz ( table 4 ) , and 4000 , 6000 , and 8000 hz ( table 5 ) . for comparing the mean hearing thresholds of each age band , the independent variable was the average hearing thresholds and the between factor was the age band . results of mean thresholds of 500 , 1000 , and 2000 hz revealed that there were significant differences among the age bands for males [ f(4 , 213)=12.042 , p<0.001 ] and for females [ f(4 , 293)=15.760 , p<0.001 ] . results of mean thresholds of 4000 , 6000 , and 8000 hz also showed that there were significant differences among the age bands for males [ f(4 , 213)=12.478 , p=0.000 ] and for females [ f(4 , 293)=15.760 , p=0.000 ] . table 6 and table 7 provide summary of post hoc tests for mean thresholds of 500 , 1000 , 2000 hz and those of 4000 , 6000 , 8000 hz , respectively . table 8 presents the mean alpha coefficient values for the present study and those for iso 7029 at each signal frequency for males and females . figs . 1 and 2 show the mean hearing thresholds of each age band for the males and females . since iso 7029 does not provide hearing thresholds according to age bands , the median hearing thresholds at each age were computed by the equation and means of the hearing thresholds for each age band were calculated for comparison . results revealed that the elderly korean population had worse hearing thresholds than the estimated hearing thresholds based on the iso 7029 equation applied to the same age band . in particular , korean males had similar hearing thresholds at high frequencies and worse hearing thresholds at low frequencies than those based on the iso 7029 . korean females had worse hearing thresholds at all frequencies than those based on the iso 7029 . table 2 lists the numerical data of hearing threshold distributions for the male and female participants . additionally , figs . these patterns were consistent with iso 7029 as well as with those in previous studies . a previous study showed that significant differences between genders occurred between 3000 and 8000 hz . in the present study , hearing thresholds across age bands in males were significantly higher than those in females from 1000 to 8000 hz . we also found that pure - tone threshold averages ( ptas ) of low ( 500 , 1000 , and 2000 hz ) and high frequencies ( 4000 , 6000 , and 8000 hz ) were gradually elevated in males as compared to those in females with increasing age . again , greater pta differences in high frequencies were observed in males than in females . this excessive noise exposure is one of the main reasons of elevated hearing - frequency hearing loss . most korean males of 60 years and above were required to serve in the military for at least 21 months . recent studies have reported that noise - induced hearing loss and tinnitus were increased due to noise exposure during military service . therefore , it is possible that exposure to excessive noise during military service affected the elevated hearing sensitivity of korean males . . taken together , these results suggest a greater amount of age - related hearing loss in males than in females and more hearing loss at higher frequencies than at lower frequencies . compared with the alpha coefficients of the iso 7029 , the alpha coefficients of the present study were higher for both genders except for high frequencies in males . in particular , the alpha coefficients of the present study for both males and females at low frequencies were higher than those of the iso 7029 . these results are consistent with previous studies . in the study by wiley , et al . , the hearing sensitivity for individuals aged 48 - 65 years was compared with the iso 7029 median thresholds for males and females . the results revealed that the expected hearing thresholds based on the iso 7029 were better than those in the previous data . the differences between the data and the iso values were larger for males than for females . in the study by stenklev and laukli , alpha coefficients were significantly higher than the iso values , especially at 125 - 4000 hz , for females . our results also indicated that the differences between the data and the iso values were larger for females across all frequencies . table 2 lists the numerical data of hearing threshold distributions for the male and female participants . additionally , figs . these patterns were consistent with iso 7029 as well as with those in previous studies . a previous study showed that significant differences between genders occurred between 3000 and 8000 hz . in the present study , hearing thresholds across age bands in males were significantly higher than those in females from 1000 to 8000 hz . we also found that pure - tone threshold averages ( ptas ) of low ( 500 , 1000 , and 2000 hz ) and high frequencies ( 4000 , 6000 , and 8000 hz ) were gradually elevated in males as compared to those in females with increasing age . again , greater pta differences in high frequencies were observed in males than in females . this excessive noise exposure is one of the main reasons of elevated hearing - frequency hearing loss . most korean males of 60 years and above were required to serve in the military for at least 21 months . recent studies have reported that noise - induced hearing loss and tinnitus were increased due to noise exposure during military service . therefore , it is possible that exposure to excessive noise during military service affected the elevated hearing sensitivity of korean males . furthermore , the alpha coefficients were larger at high frequencies than at low frequencies . taken together , these results suggest a greater amount of age - related hearing loss in males than in females and more hearing loss at higher frequencies than at lower frequencies . compared with the alpha coefficients of the iso 7029 , the alpha coefficients of the present study were higher for both genders except for high frequencies in males . in particular , the alpha coefficients of the present study for both males and females at low frequencies were higher than those of the iso 7029 . these results are consistent with previous studies . in the study by wiley , et al . , the hearing sensitivity for individuals aged 48 - 65 years was compared with the iso 7029 median thresholds for males and females . the results revealed that the expected hearing thresholds based on the iso 7029 were better than those in the previous data . the differences between the data and the iso values were larger for males than for females . in the study by stenklev and laukli , alpha coefficients were significantly higher than the iso values , especially at 125 - 4000 hz , for females . our results also indicated that the differences between the data and the iso values were larger for females across all frequencies . in conclusion , we presented the data of hearing thresholds for a korean screened population aged from 60 - 84 . it is difficult to compare our data directly with those of previous studies due to differences in sample size , age range , and etc . we conclude that 1 ) hearing threshold sensitivity increases with age , 2 ) greater hearing losses at high frequencies appear across all age groups , and 3 ) greater hearing losses are seen in males than in females . as discussed above , these data will be used for establishing a new korean standard with data from 2003 and for updating the iso 7029 which is presently under revision .
neurofibromatosis type 1 ( nf1 ) is a genetic neurocutaneous disorder with multisystem manifestations , including a predisposition to tumor formation and bone dysplasias . studies over the last decade have shown that nf1 can also be associated with significant motor deficits , such as poor coordination , low muscle tone , and easy fatigability . these have traditionally been ascribed to developmental central nervous system and cognitive deficits . however , recent preclinical studies have also illustrated a primary role for the nf1 gene product in muscle growth and metabolism ; these findings are consistent with clinical studies demonstrating reduced muscle size and muscle weakness in individuals with nf1 . currently there is no evidence - based intervention for nf1 muscle and motor deficiencies ; this review identifies key research areas where improved mechanistic understanding could unlock new therapeutic options .	neurofibromatosis type 1 ( nf1 ) is an autosomal dominant genetic disorder that affects approximately 1 in 3000 births . individuals with nf1 are predisposed to developing abnormalities in a number of body systems ; individually and cumulatively they can have a significant impact on quality of life . for example , skeletal deformities such as scoliosis and pseudarthroses , are uncommon but can be associated with considerable morbidity . benign , malignant , and disfiguring tumors ( termed neurofibromas ) are characteristic of the condition and can be challenging to manage . neuropsychological impairments in nf1 , such as executive dysfunction , inattention , specific learning disorder and reduced social competency , can result in reduced social participation and social isolation . the clinical diagnosis of nf1 relies on fulfilling at least two of the seven diagnostic criteria ; caf au lait macules , skinfold freckling , neurofibromas , lisch nodules , optic pathway tumors , bone dysplasia or a family history . notably , some of these features are congenital in origin , while some manifest over time at characteristic developmental stages . skeletal muscle and motor deficits , such as reduced muscle size , muscle weakness , and poor co - ordination however , recent preclinical and clinical studies have indicated a primary role for the nf1 gene product , neurofibromin , in muscle growth and metabolism . neurofibromin has been characterized as a gtpase activating protein that is a negative regulator of the ras gtpase . loss of functional neurofibromin leads to a dose - dependent increase in ras signaling , which can profoundly affect cell proliferation , differentiation , and function [ reviewed in . in this review we will discuss the evidence for neurological and cognitive deficits contributing to the motor / muscle phenotype . this review also refocuses discussion on previous studies that describe a vital role for nf1 in skeletal muscle development , as well as the emerging pre - clinical models that suggest a novel regulatory role for nf1 in muscle metabolism . children with nf1 have been shown to have a generalized decrease in intellectual function ( mean iq in the low 90 s ) as well as a higher rate of specific cognitive deficits ( i.e. attention , visuoperceptual skills , language and executive function ) . neurocognitive studies have also frequently reported impairment in motor abilities in individuals with nf1 ( table 1 ) , including mild impairment in gross and fine motor tasks[14 - 16 ] and impairment on the beery test of visuomotor integration , which requires subjects to draw increasingly difficult shapes and figures[17 - 19 ] . individuals with nf1 also present with deficits in a range of functional tasks that likely have significant impact on quality of life . for example , significant impairment in balance , muscle strength , and upper limb co - ordination in nf1 has been observed using the bot-2 test of motor proficiency . mild to moderate deficits in manual dexterity , balance , and ball handling skills , deficient motor timing and reaction time , reduced fine motor speed , and impaired handwriting in nf1 children has also been reported . gait assessment was performed in 46 children and adolescents with nf1 ( ages 7 - 17yrs ) using the gaitrite electronic walkway , and results were compared with a battery of cognitive tests , including the wechsler intelligence scale for children and sub - tests from the cambridge automated neuropsychological test battery . the largest correlations were found between deficits in gait width and spatial working memory ( r=0.594 , p<0.01 ) , and deficits in running speed and agility with impaired strategy generation ( r=0.549 , p<0.01 ) , supporting speculation of a link between nf1 motor deficits and abnormal central nervous system ( cns ) function . while the mechanisms of interaction between the cns and peripheral nervous system ( pns ) and motor dysfunction are unclear , structural and functional brain abnormalities are a feature of the condition that may contribute to motor impairments . neurofibromin plays a significant role in the developing brain , with loss of expression resulting in increased cell proliferation and differentiation , ultimately impacting morphology . a recurrent finding has been an increase in total brain volume , involving both grey and white matter . diffusion weighted magnetic resonance imaging ( mri ) in individuals with nf1 indicates reduced white matter integrity in a number of regions closely linked to motor function . these include the : ( 1 ) corpus callosum , ( 2 ) caudate nucleus ; a sub - region of the basal ganglia involved in goal - direct behavior and voluntary movement , and ( 3 ) thalamus ; a sub - cortical nuclear complex that receives and relays cortical and sub - cortical inputs that sub - serve sensory and motor mechanisms , as well as cognitive abilities . volumetric studies report these structures as abnormally large in nf1 cohorts , suggesting a reduced signal - to - noise ratio . moore et al have further shown significant associations between increased corpus callosum size and reduced motor performance in children with nf1 . there is also mounting evidence for functional abnormalities within the thalamus and caudate in individuals with nf1 . both adult and pediatric positron emission tomography studies report thalamic hypometabolism , suggesting reduced thalamic signal processing , and a recent event - related functional mri study identified abnormal right caudate activation during a spatial working memory task ; a cognitive ability significantly associated with impaired gait in nf1 . individuals with nf1 also commonly show focal areas of high t2 signal intensity on mri , which have been associated with reduced fine motor skill . mechanistically , one clinical study has examined sensory and motor neuropathy in nf1 as possibly contributory . electrophysiological measures in 39 individuals with nf1 aged 10 - 56 revealed motor polyneuropathy was a rare manifestation , and while abnormalities in multimodal evoked potentials ( visual , auditory and sensory ) were seen commonly , many were associated with tumors or lesions . further clinical studies correlating neurological assessments with strength and other motor outcomes are needed to define the influence of the cns and pns in the nf1 motor phenotype . while preclinical mouse models have been used to investigate mechanisms underlying some neurocognitive deficits , a relationship between cognition and motor performance has not yet been defined in these systems . abnormalities such as the learning and attention deficits have been extensively examined in the nf1 mouse[38 - 43 ] . for example , increased gaba release in the hippocampus has been shown to underlie learning deficits , and can be rescued by inhibition of erk signaling , while lovastatin treatment has been shown to rescue deficits in learning and attention . however , interactions between these deficits and motor function remain unknown . the most significant motor deficit seen in this mouse line was impaired grip strength using a hanging wire test , and there was no demonstration of a neurocognitive basis for this result . furthermore , this line failed to show any deficiencies in motor performance tests linked to cerebellar function . decreased dopamine levels in the striatum have also been identified in a further nf1 mouse model with bi - allelic nf1 inactivation in glia . these mice exhibited reduced exploratory behaviors as well as selective and non - selective attention abnormalities , which were rescued by pharmacologic intervention to restore dopamine levels . while speculative , abnormal dopamine levels may underlie some of the motor impairments observed in individuals with nf1 . indeed some gait characteristics identified in a pediatric nf1 cohort , including a shorter step length and longer step time , resemble those seen in early parkinson disease ; a disorder associated with reduced dorsal striatal dopamine levels . further insight into the interactions between neurocognitive development and motor deficits may come from more advanced mouse models of conditional double inactivation of nf1 in neurons . in 2005 , stevenson et al . published data from an analysis of 40 individuals with nf1 using peripheral quantitative computed tomography ( pqct ) scanning . individuals with nf1 presented with a significant reduction in muscle cross - sectional area compared to age matched controls . comparative findings were seen in a pediatric nf1 cohort where lean tissue was measured using dual - energy x - ray absorptiometry ( dexa ) . children with nf1 had a significantly reduced lean tissue mass . while reduced muscle size may imply a reduction in strength , muscle functional outcomes were not assessed in these primarily radiographic studies . however , in a seminal 2009 study , souza et al performed hand grip dynamometry testing of 21 subjects ( age 7 - 60 yrs ) with nf1 compared to gender , aged , and physical activity matched controls , and demonstrated a significant reduction in grip strength in the nf1 cohort . findings of muscle functional impairment in nf1 have been subsequently reported in clinical studies ( table 2 ) . reduced strength of the hip extensor muscles has been described using hand held dynamometry . likewise , a 2013 trial investigating lower body muscle function in nf1 children , found that jumping force ( n / kg ) and jumping power ( w / kg ) were both significantly reduced . a cohort of 17 individuals with nf1 , along with gender , age , and bodyweight matched control subjects , underwent maximal oxygen consumption ( vo2 max ) testing , a measure of maximal aerobic exercise capacity . individuals with nf1 had a reduced vo2 max as well as a reduced maximal systolic blood pressure . while the authors acknowledged that there was some difficulty in recruiting activity matched controls , it remains to be thoroughly investigated whether reduced physical activity accounts for reduced exercise capacity in nf1 . given the mounting evidence for motor and muscular deficits in nf1 , continued exercise studies of this kind are warranted , particularly those assessing quality of life outcome measures . muscle size and muscle function in nf1 . insight gained from research into nf1 muscle function may be applicable to other related genetic diseases . nf1 belongs to the rasopathy family of diseases , which includes costello syndrome , cardiofaciocutaneous syndrome , and noonan syndrome . in a 2012 clinical study , reduced grip strength and muscle weakness was identified as a common feature of them all . it is unclear whether these conditions share a common mechanism for muscle weakness downstream of altered ras signaling . however , if this is the case , any successful pathway - targeted interventions that improve nf1 muscle performance , may have broad clinical applicability to other rasopathies . early evidence suggesting a critical role for nf1 in skeletal muscle development came from in vitro experiments assessing gene expression during myoblast differentiation . levels of nf1 mrna , and neurofibromin were elevated during differentiation , and a concomitant decrease in activated p21-ras was observed . double inactivation of nf1 was found to be embryonically lethal , and nf1 null embryos showed underdeveloped cardiac and skeletal muscle . however , it remained unclear whether these effects on muscle were secondary to some other failure in the developmental program , and it was nt until recently that a key role for nf1 in muscle was demonstrated in an animal model . developed a limb - specific nf1 knockout mouse ( nf1prx1 ) using a prx1-cre transgene to drive deletion of nf1 in cells of the mesenchymal lineage . furthermore , consistent with in vitro data , analysis of mutant embryos revealed hyperactive ras / mapk signaling , and impaired myoblast differentiation in the developing limbs . while the nf1prx1-/- mouse model demonstrated the requirement of nf1 for normal limb muscle development , prx1-cre driven recombination is not restricted to the muscle lineage . thus , the inactivation of nf1 in other mesenchymal tissues including adipocytes , connective tissue , and bone , were potential confounders to interpretation . to overcome this , a skeletal muscle specific nf1 knockout mouse ( nf1myod ) was subsequently generated , by crossing the myod - cre transgenic mouse with the nf1-flox line . homozygous nf1 inactivation in muscle was lethal in the first week of life , and while pups were born at normal bodyweight , they had stunted growth and a high rate of maternal infanticide was observed . electron microscopy ( em ) imaging of nf1myod muscle specimens showed no evidence of cytoarchitectural abnormalities , including protein aggregates , myofibrillar disruption , or z - line streaming . while nf1prx1-/- muscle has been described as dystrophic , this may be a misnomer , as neither mouse model nor patient muscle biopsies have been characterized as having progressive loss of cytoskeletal or membrane protein integrity . em of 3-day old nf1myod muscle samples unexpectedly revealed excessive accumulations of intramyocellular lipid , which was subsequently confirmed by oil red o staining . this led to speculation that nf1 may have a key role in the regulation of muscle lipid metabolism . analysis of adult nf1prx1-/- muscle samples revealed similarly elevated triglyceride levels , 10-fold that of controls . increased fatty acid synthesis may underlie these accumulations in the nf1prx1-/- mice , as a substantive increase in the expression of fatty acid synthase was observed . metabolic dysregulation was also seen for a range of mitochondrial enzymes , including succinate dehydrogenase ( sdh ) , -hydroxyacyl - coa dehydrogenase ( bhad ) , and medium - chain acyl - coa dehydrogenase ( mcad ) . the expression of mitochondrial fatty acid transport protein carnitine palmitoyl transferase-1 ( cpt-1 ) , and membrane transport proteins , cd36 , and fatty acid transport protein 4 ( fatp4 ) were also reduced . for example , it is difficult to separate any primary deficits responsible , from any downstream or compensatory metabolic changes in the mature nf1prx1-/- muscle . interestingly , analysis of neonatal nf1myod muscle showed only the intramyocellular lipid phenotype , suggesting that lipid accumulation may be the initiating factor , and that subsequent molecular and metabolic dysregulation is temporal in nature . although the precise mechanisms remain unknown , these mouse data demonstrate a novel metabolic regulatory role for neurofibromin in muscle . one possibility is that nf1 muscle has commonalities with the lipid storage myopathies ( lsms ) , which also present with progressive muscle weakness and muscle lipid accumulation . if parallels are found to exist with the lsms , this may provide insight into potential interventions for nf1 . for example , primary carnitine deficiency ( pcd ) leads to an impairment of lipid transport into the mitochondria , a resultant accumulation of lipid droplets , and muscle weakness . pcd patients have been successfully treated with high dose l - carnitine supplementation . while such speculation is attractive to entertain , evidence for lipid accumulation in human nf1 samples has not been firmly established . identifying lipid accumulation in human nf1 muscle biopsies will be an important goal for researchers in order to demonstrate the relevance of these murine models . a number of historical and recent studies raise important questions regarding the role of the nf1 gene in muscle development and function . in the 1990s , gutmann et al identified cardiac and skeletal muscle isoforms of nf1 . to date , the functional importance of these isoforms remains unclear . it is possible that they have a unique role in the regulation of muscle development and/or metabolism . isoform - specific knockout models may be able to provide insight into the role of alternatively spliced variants of neurofibromin in muscle . furthermore , the genetics of nf1 in muscle are yet to be elucidated . some manifestations of nf1 are associated with heterozygosity ( haploinsufficiency ) and others with double inactivation . for example , local double inactivation has been observed in nf1 tumors as well as in tibial pseudarthrosis tissue . to date , no studies have addressed the potential double inactivation of nf1 in muscle . myofibers are multinucleated cells where sporadic double inactivation in individual nuclei could have unpredictable effects . analyzing double inactivation in myofibers may be challenging , but fluorescent in situ hybridization for nf1 on human muscle biopsies could be a feasible approach . one confounding factor in interpretation of clinical data is that the cognitive , motor control and psycho - social effects of nf1 may indirectly influence physical activity . recent data indicates that children with nf1 have reduced participation in formal and informal physical activities . finding ways to accommodate this into both studies of the underlying biological weakness and strategies for exercise - based intervention may have its own challenges . one intriguing possibility is that nf1 mutations and associated muscle weakness may increase the risk or severity of other related or unrelated conditions . for example , spinal complications such as scoliosis can be a major source of morbidity in nf1 . late onset scoliosis has been described in adolescents with nf1 with no underling bone abnormalities , and it is possible that weakness or hypotonia may be contributory . in addition , two recent case reports suggest the potential for interactions between nf1 and other muscle - related genetic conditions . one report describes an individual with digeny for mutations in nf1 and ryanodine receptor 1 , resulting in myopathy . the other shows a previously unreported mutation in an nf1 locus leading to mitochondrial complex i deficiency , hypotonia , and developmental delay . continued identification of clinical presentations of this kind may provide useful insight . from a therapeutic standpoint , it will be critical to ascertain the capacity of exercise regimes to modify the muscle and motor phenotypes in nf1 . the current literature is limited to a single case study that reports improved jumping and throwing performance in children with nf1 , following a plyometric training program . however , this study was small ( n=3 ) , the children were of variable ages and genders , and the study lacked a control cohort . larger randomized and controlled exercise intervention studies are greatly needed to answer questions regarding the effects of exercise training on motor control , muscle size and strength , fatigue , and quality of life outcomes in individuals with nf1 . while physical therapies are often favored if they can produce significant benefits , pathway - specific pharmacological interventions remain a potential treatment for those found unresponsive to exercise . the ras - mek - erk pathway is the canonical pathway in nf1 , but this signaling cascade is also recognized for its role in muscle . constitutively active mek has been shown to directly bind and repress myogenic transcription factors , inhibiting myogenic differentiation in vitro . furthermore , in a cancer setting , mek / erk inhibition has been shown to be anabolic for skeletal muscle in humans and mice . this pathway is likely to be of particular relevance for interventions aiming to improve muscle function in nf1 . souza et al . can be credited for initiating an explosion of activity in the field of nf1 muscle research in 2009 . since then , a range of clinical studies have confirmed reduced motor performance and/or muscle impairment in individuals with nf1 . while there are associations between neurological abnormalities and the nf1 muscle / motor phenotype , the mechanisms underlying these interactions are yet to be elucidated , and remain an area for further research . mechanistically , recent studies using genetically modified mouse models have provided strong evidence for a metabolic regulatory role for neurofibromin in muscle , likely contributing to the phenotype . this review has also reflected on a number of historically overlooked or potentially undervalued studies . the key roles for nf1 in muscle development are preceded by studies showing increases in nf1 gene and protein expression during myogenic differentiation . the findings of deficits in muscle strength are similarly preceded by radiographic studies showing decreases in muscle mass . however , it is the studies describing muscle - specific nf1 isoforms that are perhaps the most relevant to revisit , as these isoforms may have as yet undefined roles in the nf1-muscle phenotype . in summary , there have been significant advances in our understanding due to a co - ordination of basic and clinical research studies .
mosquitoes act as a vector for most of the life threatening diseases like malaria , yellow fever , dengue fever , chikungunya ferver , filariasis , encephalitis , west nile virus infection , etc . under the integrated mosquito management ( imm ) , emphasis was given on the application of alternative strategies in mosquito control . the continuous application of synthetic insecticides causes development of resistance in vector species , biological magnification of toxic substances through the food chain and adverse effects on environmental quality and non target organisms including human health . application of active toxic agents from plant extracts as an alternative mosquito control strategy was available from ancient times . these are non - toxic , easily available at affordable prices , biodegradable and show broad - spectrum target - specific activities against different species of vector mosquitoes . in this article , the current state of knowledge on phytochemical sources and mosquitocidal activity , their mechanism of action on target population , variation of their larvicidal activity according to mosquito species , instar specificity , polarity of solvents used during extraction , nature of active ingredient and promising advances made in biological control of mosquitoes by plant derived secondary metabolites have been reviewed .	mosquitoes can transmit more diseases than any other group of arthropods and affect million of people throughout the world . who has declared the mosquitoes as mosquito borne diseases are prevalent in more than 100 countries across the world , infecting over 700,000,000 people every year globally and 40,000,000 of the indian population . they act as a vector for most of the life threatening diseases like malaria , yellow fever , dengue fever , chikungunya ferver , filariasis , encephalitis , west nile virus infection , etc . , in almost all tropical and subtropical countries and many other parts of the world . to prevent proliferation of mosquito borne diseases and to improve quality of environment and public health the major tool in mosquito control operation is the application of synthetic insecticides such as organochlorine and organophosphate compounds . but this has not been very successful due to human , technical , operational , ecological , and economic factors . in recent years , use of many of the former synthetic insecticides in mosquito control programme has been limited . it is due to lack of novel insecticides , high cost of synthetic insecticides , concern for environmental sustainability , harmful effect on human health , and other non - target populations , their non biodegradable nature , higher rate of biological magnification through ecosystem , and increasing insecticide resistance on a global scale23 . thus , the environmental protection act in 1969 has framed a number of rules and regulations to check the application of chemical control agents in nature4 . it has prompted researchers to look for alternative approaches ranging from provision of or promoting the adoption of effective and transparent mosquito management strategies that focus on public education , monitoring and surveillance , source reduction and environment friendly least - toxic larval control . these factors have resulted in an urge to look for environment friendly , cost - effective , biodegradable and target specific insecticides against mosquito species . considering these , the application of eco - friedly alternatives such as biological control of vectors has become the central focus of the control programmme in lieu of the chemical insecticides . one of the most effective alternative approaches under the biological control programme is to explore the floral biodiversity and enter the field of using safer insecticides of botanical origin as a simple and sustainable method of mosquito control . further , unlike conventional insecticides which are based on a single active ingredient , plant derived insecticides comprise botanical blends of chemical compounds which act concertedly on both behavourial and physiological processes . identifying bio - insecticides that are efficient , as well as being suitable and adaptive to ecological conditions , is imperative for continued effective vector control management . botanicals have widespread insecticidal properties and will obviously work as a new weapon in the arsenal of synthetic insecticides and in future may act as suitable alternative product to fight against mosquito borne diseases . roark5 described approximately 1,200 plant species having potential insecticidal value , while sukumar et al6 listed and discussed 344 plant species that only exhibited mosquitocidal activity . shallan et al in 20057 reviewed the current state of knowledge on larvicidal plant species , extraction processes , growth and reproduction inhibiting phytochemicals , botanical ovicides , synergistic , additive and antagonistic joint action effects of mixtures , residual capacity , effects on non - target organisms , resistance and screening methodologies , and discussed some promising advances made in phytochemical research . table i summarized the mosquitocidal activities of various herbal products from edible crops , ornamental plants , trees , shrubs , herbs , grasses and marine plants according to the exaction procedure developed in eleven different solvent systems and the nature of mosquitocidal activities against different life stages of different vector species as a ready reference for further studies . . applications of phytochemicals in mosquito control were in use since the 1920s8 , but the discovery of synthetic insecticides such as ddt in 1939 side tracked the application of phytochemicals in mosquito control programme . after facing several problems due to injudicious and over application of synthetic insecticides in nature , re - focus on phytochemicals that are easily biodegradable and have no ill - effects on non - target organisms was appreciated . since then , the search for new bioactive compounds from the plant kingdom and an effort to determine its structure and commercial production has been initiated . at present phytochemicals botanicals are basically secondary metabolites that serve as a means of defence mechanism of the plants to withstand the continuous selection pressure from herbivore predators and other environmental factors . several groups of phytochemicals such as alkaloids , steroids , terpenoids , essential oils and phenolics from different plants have been reported previously for their insecticidal activities7 . insecticidal effects of plant extracts vary not only according to plant species , mosquito species , geographical varities and parts used , but also due to extraction methodology adopted and the polarity of the solvents used during extraction . a wide selection of plants from herbs , shrubs and large trees was used for extraction of mosquito toxins . phytochemicals were extracted either from the whole body of little herbs or from various parts like fruits , leaves , stems , barks , roots , etc . , of larger plants or trees . in all cases where the most toxic substances were concentrated upon , found and extracted for mosquito control . more than 2000 plant species have been known to produce chemical factors and metabolites of value in pest control programmes . members of the plant families- solanaceae , asteraceae , cladophoraceae , labiatae , miliaceae , oocystaceae and rutaceae have various types of larval , adulticidal or repellent activities against different species of mosquitoes7 . human beings have used plant parts , products and secondary metabolites of plant origin in pest control since early historical times . vector control has been practiced since the early 20th century . during the pre - ddt era , reduction of vector mosquitoes mainly depended on environmental management of breeding habitats , i.e. , source reduction . during that period , some botanical insecticides used in different countries were chrysanthemum , pyrethrum , derris , quassia , nicotine , hellebore , anabasine , azadirachtin , d - limonene camphor , turpentine , etc7 . from the early 1950s , ddt and other synthetic organochloride and organophosphate insecticides were extensively used to interrupt transmission of vector borne diseases by reducing densities , human - vector contact and , in particular , the longevity of vector mosquitoes . in the mid-1970s , the resurgence of vector borne diseases , along with development of insecticide resistance in vector population , poor human acceptance of indoor house spraying and environmental concerns against the use of insecticides led to a rethinking in vector control strategies10 . as a result , emphasis was given on the application of alternative methods in mosquito control as part of the integrated mosquito management ( imm)11 . integrated mosquito management ( imm ) is a decision - making process for the management of mosquito populations , involving a combination of methods and strategies for long - term maintenance of low levels of vectors . the purpose of imm is to protect public health from diseases transmitted by mosquitoes , maintain healthy environment through proper use and disposal of pesticides and improve the overall quality of life through practical and effective pest control strategies . the main approaches of imm include : ( i ) source reduction and habitat management by proper sanitation , water management in temporary and permanent water bodies , and channel irrigation . vegetation management is also necessary to eliminate protection and food for mosquito larvae ; ( ii ) larviciding by application of dipteran specific bacteria , insect growth regulators , surface films and oils , expanded polystyrene beads , phytochemicals , organophosphates and organochlorides , ( iii ) adulticiding by application of synthetic pyrethroids , organophosphates and synthetic or plant derived repellents , insecticide impregnated bed nets , genetic manipulations of vector species , etc . , ( iv ) use of mosquito density assessment in adult and larval condition and disease surveillance ; and ( v ) application of biological control methods by using entomophagous bacteria , fungi , microsporidians , predators and parasites . of the above avenues of imm , larviciding approach is the more proactive , proenvironment , target specific and safer approach than controlling adult mosquitoes . application of larvicide from botanical origin was extensively studied as an essential part of imm , and various mosquito control agents such as ocimenone , rotenone , capllin , quassin , thymol , eugenol , neolignans , arborine and goniothalamin were developed7 . the efficacy of phytochemicals against mosquito larvae can vary significantly depending on plant species , plant parts used , age of plant parts ( young , mature or senescent ) , solvent used during extraction as well as upon the available vector species . sukumar et al6 have described the existence of variations in the level of effectiveness of phytochemical compounds on target mosquito species vis - - vis plant parts from which these were extracted , responses in species and their developmental stages against the specified extract , solvent of extraction , geographical origin of the plant , photosensitivity of some of the compounds in the extract , effect on growth and reproduction . changes in the larvicidal efficacy of the plant extracts occurred due to geographical origin of the plant ( in citrus sp18396465 , jatropha sp132021 , ocimum sanctum22356582 , momordica charantia222449 , piper sp54638995 and azadirechta indica65 ) ; response in the different mosquito species ( in curcuma domestica26 , withania somnifera13 , jatropha curcas1320 , piper retrofractum63 , cestrum diurnum58 , citrullus vulgaris5071 , and tridax procumbens3031 ) ; due to variation in the species of plant examined ( in euphorbia sp22283751 , phyllanthus sp20 , curcuma sp36 , solanum sp16295760757996 , ocimum sp23356582 , eucalyptus sp22283751 , plumbago sp20 , vitex sp5093 , piper sp54638995 , annona sp485469 , and cleome sp3178 ) and between plant parts used to study the larvicidal efficacy ( in euphorbia tirucalli2851 , solanum xanthocarpum16 , azadirechta indica65 , solanum villosum57607996 , annona squamosa485469 , withania somnifera13 , melia azedarach45 , moringa oleifera46 and ocimum sanctum3582 ) . however , the principal objective of the present documentation is to report the changes in larvicidal potentiality of the plant extracts due to change of the particular solvent used during extraction . variation of the larvicidal potential of the same plant changed with the solvents used as evidenced in case of solanum xanthocarpum16 , euphorbia tirucalli2851 , momordica charantia222449 , eucalyptus globules14152883 , citrullus colocynthis13 , azadirechta indica65 , annona squamosa485469 and solanum nigrum2975 . it has been shown that the extraction of active biochemical from plants depends upon the polarity of the solvents used . polar solvent will extract polar molecules and non - polar solvents extract non - polar molecules . this was achieved by using mainly eleven solvent systems ranging from hexane/ petroleum ether , the most non polar ( polarity index of 0.1 that mainly extracts essential oil ) to that of water , the most polar ( polarity index of 10.2 ) that extracts biochemical with higher molecular weights such as proteins , glycans , etc . chloroform or ethyl acetate are moderately polar ( polarity index of 4.1 ) that mainly extracts steroids , alkaloids , etc . it has been found that in most of the studies solvent with minimum polarity have been used such as hexane or petroleum ether or that with maximum polarity such as aqueous/ steam distillation . however , those biochemical that were extracted using moderately polar solvents were also seen to give good results as reported by a few bioassay . thus , different solvent types can significantly affect the potency of extracted plant compounds and there is difference in the chemo - profile of the plant species . in table i , the lowest lc50 value was reported in solenostemma argel against cx . several other plants such as nyctanthes arbotristis38 , atlantia monophylla57 , centella asiatica40 , cryptotaenia paniculata76 were also reported with promising lc50 values . these extracts may be fractioned in order to locate the particular bioactive toxic agent responsible for larval toxicity . table i also reported that most of the studies were carried out on culex mosquitoes and aedes was the least frequently chosen mosquitoes for all the experiments . in several studies , instead of a particular solvent , combination of solvents or serial extraction by different solvents according to their polarity has also been tried and good larvicidal potentiality found as a result96 . the plant world comprises a rich untapped pool of phytochemicals that may be widely used in place of synthetic insecticides in mosquito control programme . kishore et al97 reviewed the efficacy of phytochemicals against mosquito larvae according to their chemical nature and described the mosquito larvicidal potentiality of several plant derived secondary materials , such as , alkanes , alkenes , alkynes and simple aromatics , lactones , essential oils and fatty acids , terpenes , alkaloids , steroids , isoflavonoids , pterocarpans and lignans . they also documented the isolation of several bioactive toxic principles from various plants and reported their toxicity against different mosquito species ( table ii ) . identification of various bioactive toxic principles from plant extract and their relative mosquitocidal efficacy generally the active toxic ingredients of plant extracts are secondary metabolites that are evolved to protect them from herbivores . the insects feed on these secondary metabolites potentially encountering toxic substances with relatively non - specific effects on a wide range of molecular targets . these targets range from proteins ( enzymes , receptors , signaling molecules , ion - channels and structural proteins ) , nucleic acids , biomembranes , and other cellular components98 . this in turn , affects insect physiology in many different ways and at various receptor sites , the principal of which is abnormality in the nervous system ( such as , in neurotransmitter synthesis , storage , release , binding , and re - uptake , receptor activation and function , enzymes involved in signal transduction pathway)98 . rattan98 reviewed the mechanism of action of plant secondary metabolites on insect body and documented several physiological disruptions , such as inhibition of acetylecholinestrase ( by essential oils ) , gaba - gated chloride channel ( by thymol ) , sodium and potassium ion exchange disruption ( by pyrethrin ) and inhibition of cellular respiration ( by rotenone ) . such disruption also includes the blockage of calcium channels ( by ryanodine ) , of nerve cell membrane action ( by sabadilla ) , of octopamine receptors ( thymol ) , hormonal balance disruption , mitotic poisioning ( by azadirachtin ) , disruption of the molecular events of morphogenesis and alteration in the behaviour and memory of cholinergic system ( by essential oil ) , etc . of these , the most important activity is the inhibition of acetylcholinerase activity ( ache ) as it is a key enzyme responsible for terminating the nerve impulse transmission through synaptic pathway ; ache has been observed to be organophosphorus and carbamate resistant , and it is well - known that the alteration in ache is one of the main resistance mechanisms in insect pests99 . several studies have documented the efficacy of plant extracts as the reservoier pool of bioactive toxic agents against mosquito larvae . but only a few have been commercially produced and extensively used in vector control programmes . the main reasons behind the failure in laboratory to land movements of bioactive toxic phytochemicals are poor characterization and inefficiency in determining the structure of active toxic ingredients responsible for larvicidal activity . for the production of a green biopesticide , the following steps can be recommended during any research design with phytochemicals : ( i ) screening of floral biodiversity in search of crude plant extracts having mosquito larvicidal potentiality ; ( ii ) preparation of plant solvent extracts starting from non - polar to polar chemicals and determination of the most effective solvent extract ; ( iii ) evaporation of the liquid solvent to obtain solid residue and determination of the lethal concentration ( lc50/lc100 values ) ; ( iv ) phytochemical analysis of the solid residue and application of column chromatography and thin layer chromatography to purify and isolate toxic phytochemical with larvicidal potentiality ; ( v ) determination of the structure of active principle by infra red ( ir ) spectroscopic , nuclear magnetic resonance ( nmr ) and gas chromatography and mass spectroscopy ( gcms ) analysis ; ( vi ) study of the effect of active ingredient on non target organisms ; and ( vii ) field evaluation of the active principle before its recommendation in vector control programme and commercial production . human beings have used plant parts , products and secondary metabolites of plant origin in pest control since early historical times . vector control has been practiced since the early 20th century . during the pre - ddt era , reduction of vector mosquitoes mainly depended on environmental management of breeding habitats , i.e. , source reduction . during that period , some botanical insecticides used in different countries were chrysanthemum , pyrethrum , derris , quassia , nicotine , hellebore , anabasine , azadirachtin , d - limonene camphor , turpentine , etc7 . from the early 1950s , ddt and other synthetic organochloride and organophosphate insecticides were extensively used to interrupt transmission of vector borne diseases by reducing densities , human - vector contact and , in particular , the longevity of vector mosquitoes . in the mid-1970s , the resurgence of vector borne diseases , along with development of insecticide resistance in vector population , poor human acceptance of indoor house spraying and environmental concerns against the use of insecticides led to a rethinking in vector control strategies10 . as a result , emphasis was given on the application of alternative methods in mosquito control as part of the integrated mosquito management ( imm)11 . integrated mosquito management ( imm ) is a decision - making process for the management of mosquito populations , involving a combination of methods and strategies for long - term maintenance of low levels of vectors . the purpose of imm is to protect public health from diseases transmitted by mosquitoes , maintain healthy environment through proper use and disposal of pesticides and improve the overall quality of life through practical and effective pest control strategies . the main approaches of imm include : ( i ) source reduction and habitat management by proper sanitation , water management in temporary and permanent water bodies , and channel irrigation . vegetation management is also necessary to eliminate protection and food for mosquito larvae ; ( ii ) larviciding by application of dipteran specific bacteria , insect growth regulators , surface films and oils , expanded polystyrene beads , phytochemicals , organophosphates and organochlorides , ( iii ) adulticiding by application of synthetic pyrethroids , organophosphates and synthetic or plant derived repellents , insecticide impregnated bed nets , genetic manipulations of vector species , etc . , ( iv ) use of mosquito density assessment in adult and larval condition and disease surveillance ; and ( v ) application of biological control methods by using entomophagous bacteria , fungi , microsporidians , predators and parasites . of the above avenues of imm , larviciding approach is the more proactive , proenvironment , target specific and safer approach than controlling adult mosquitoes . application of larvicide from botanical origin was extensively studied as an essential part of imm , and various mosquito control agents such as ocimenone , rotenone , capllin , quassin , thymol , eugenol , neolignans , arborine and goniothalamin were developed7 . the efficacy of phytochemicals against mosquito larvae can vary significantly depending on plant species , plant parts used , age of plant parts ( young , mature or senescent ) , solvent used during extraction as well as upon the available vector species . sukumar et al6 have described the existence of variations in the level of effectiveness of phytochemical compounds on target mosquito species vis - - vis plant parts from which these were extracted , responses in species and their developmental stages against the specified extract , solvent of extraction , geographical origin of the plant , photosensitivity of some of the compounds in the extract , effect on growth and reproduction . changes in the larvicidal efficacy of the plant extracts occurred due to geographical origin of the plant ( in citrus sp18396465 , jatropha sp132021 , ocimum sanctum22356582 , momordica charantia222449 , piper sp54638995 and azadirechta indica65 ) ; response in the different mosquito species ( in curcuma domestica26 , withania somnifera13 , jatropha curcas1320 , piper retrofractum63 , cestrum diurnum58 , citrullus vulgaris5071 , and tridax procumbens3031 ) ; due to variation in the species of plant examined ( in euphorbia sp22283751 , phyllanthus sp20 , curcuma sp36 , solanum sp16295760757996 , ocimum sp23356582 , eucalyptus sp22283751 , plumbago sp20 , vitex sp5093 , piper sp54638995 , annona sp485469 , and cleome sp3178 ) and between plant parts used to study the larvicidal efficacy ( in euphorbia tirucalli2851 , solanum xanthocarpum16 , azadirechta indica65 , solanum villosum57607996 , annona squamosa485469 , withania somnifera13 , melia azedarach45 , moringa oleifera46 and ocimum sanctum3582 ) . however , the principal objective of the present documentation is to report the changes in larvicidal potentiality of the plant extracts due to change of the particular solvent used during extraction . variation of the larvicidal potential of the same plant changed with the solvents used as evidenced in case of solanum xanthocarpum16 , euphorbia tirucalli2851 , momordica charantia222449 , eucalyptus globules14152883 , citrullus colocynthis13 , azadirechta indica65 , annona squamosa485469 and solanum nigrum2975 . it has been shown that the extraction of active biochemical from plants depends upon the polarity of the solvents used . polar solvent will extract polar molecules and non - polar solvents extract non - polar molecules . this was achieved by using mainly eleven solvent systems ranging from hexane/ petroleum ether , the most non polar ( polarity index of 0.1 that mainly extracts essential oil ) to that of water , the most polar ( polarity index of 10.2 ) that extracts biochemical with higher molecular weights such as proteins , glycans , etc . chloroform or ethyl acetate are moderately polar ( polarity index of 4.1 ) that mainly extracts steroids , alkaloids , etc . it has been found that in most of the studies solvent with minimum polarity have been used such as hexane or petroleum ether or that with maximum polarity such as aqueous/ steam distillation . however , those biochemical that were extracted using moderately polar solvents were also seen to give good results as reported by a few bioassay . thus , different solvent types can significantly affect the potency of extracted plant compounds and there is difference in the chemo - profile of the plant species . in table i , the lowest lc50 value was reported in solenostemma argel against cx . several other plants such as nyctanthes arbotristis38 , atlantia monophylla57 , centella asiatica40 , cryptotaenia paniculata76 were also reported with promising lc50 values . these extracts may be fractioned in order to locate the particular bioactive toxic agent responsible for larval toxicity . table i also reported that most of the studies were carried out on culex mosquitoes and aedes was the least frequently chosen mosquitoes for all the experiments . in several studies , instead of a particular solvent , combination of solvents or serial extraction by different solvents according to their polarity has also been tried and good larvicidal potentiality found as a result96 . the plant world comprises a rich untapped pool of phytochemicals that may be widely used in place of synthetic insecticides in mosquito control programme . kishore et al97 reviewed the efficacy of phytochemicals against mosquito larvae according to their chemical nature and described the mosquito larvicidal potentiality of several plant derived secondary materials , such as , alkanes , alkenes , alkynes and simple aromatics , lactones , essential oils and fatty acids , terpenes , alkaloids , steroids , isoflavonoids , pterocarpans and lignans . they also documented the isolation of several bioactive toxic principles from various plants and reported their toxicity against different mosquito species ( table ii ) . generally the active toxic ingredients of plant extracts are secondary metabolites that are evolved to protect them from herbivores . the insects feed on these secondary metabolites potentially encountering toxic substances with relatively non - specific effects on a wide range of molecular targets . these targets range from proteins ( enzymes , receptors , signaling molecules , ion - channels and structural proteins ) , nucleic acids , biomembranes , and other cellular components98 . this in turn , affects insect physiology in many different ways and at various receptor sites , the principal of which is abnormality in the nervous system ( such as , in neurotransmitter synthesis , storage , release , binding , and re - uptake , receptor activation and function , enzymes involved in signal transduction pathway)98 . rattan98 reviewed the mechanism of action of plant secondary metabolites on insect body and documented several physiological disruptions , such as inhibition of acetylecholinestrase ( by essential oils ) , gaba - gated chloride channel ( by thymol ) , sodium and potassium ion exchange disruption ( by pyrethrin ) and inhibition of cellular respiration ( by rotenone ) . such disruption also includes the blockage of calcium channels ( by ryanodine ) , of nerve cell membrane action ( by sabadilla ) , of octopamine receptors ( thymol ) , hormonal balance disruption , mitotic poisioning ( by azadirachtin ) , disruption of the molecular events of morphogenesis and alteration in the behaviour and memory of cholinergic system ( by essential oil ) , etc . of these , the most important activity is the inhibition of acetylcholinerase activity ( ache ) as it is a key enzyme responsible for terminating the nerve impulse transmission through synaptic pathway ; ache has been observed to be organophosphorus and carbamate resistant , and it is well - known that the alteration in ache is one of the main resistance mechanisms in insect pests99 . several studies have documented the efficacy of plant extracts as the reservoier pool of bioactive toxic agents against mosquito larvae . but only a few have been commercially produced and extensively used in vector control programmes . the main reasons behind the failure in laboratory to land movements of bioactive toxic phytochemicals are poor characterization and inefficiency in determining the structure of active toxic ingredients responsible for larvicidal activity . for the production of a green biopesticide , the following steps can be recommended during any research design with phytochemicals : ( i ) screening of floral biodiversity in search of crude plant extracts having mosquito larvicidal potentiality ; ( ii ) preparation of plant solvent extracts starting from non - polar to polar chemicals and determination of the most effective solvent extract ; ( iii ) evaporation of the liquid solvent to obtain solid residue and determination of the lethal concentration ( lc50/lc100 values ) ; ( iv ) phytochemical analysis of the solid residue and application of column chromatography and thin layer chromatography to purify and isolate toxic phytochemical with larvicidal potentiality ; ( v ) determination of the structure of active principle by infra red ( ir ) spectroscopic , nuclear magnetic resonance ( nmr ) and gas chromatography and mass spectroscopy ( gcms ) analysis ; ( vi ) study of the effect of active ingredient on non target organisms ; and ( vii ) field evaluation of the active principle before its recommendation in vector control programme and commercial production . today , environmental safety is considered to be of paramount importance . an insecticide does not need to cause high mortality on target organisms in order to be acceptable but should be eco - friedly in nature . phytochemicals may serve as these are relatively safe , inexpensive and readily available in many parts of the world . several plants are used in traditional medicines for the mosquito larvicidal activities in many parts of the world . according to bowers et al100 , the screening of locally available medicinal plants for mosquito control would generate local employment , reduce dependence on expensive and imported products , and stimulate local efforts to enhance the public health system . the ethno - pharmacological approaches used in the search of new bioactive toxins from plants appear to be predictive compared to the random screening approach . the recently developed new isolation techniques and chemical characterization through different types of spectroscopy and chromatography together with new pharmacological testing have led to an interest in plants as the source of new larvicidal compounds . synergestic approaches such as application of mosquito predators with botanical blends and microbial pesticides will provide a better effect in reducing the vector population and the magnitude of epidemiology .
during late summer 2001 in austria , a series of deaths in several species of birds occurred , similar to the beginning of the west nile virus ( wnv ) epidemic in the united states . we necropsied the dead birds and examined them by various methods ; pathologic and immunohistologic investigations suggested a wnv infection . subsequently , the virus was isolated , identified , partially sequenced , and subjected to phylogenetic analysis . the isolates exhibited 97% identity to usutu virus ( usuv ) , a mosquito - borne flavivirus of the japanese encephalitis virus group ; usuv has never previously been observed outside africa nor associated with fatal disease in animals or humans . if established in central europe , this virus may have considerable effects on avian populations ; whether usuv has the potential to cause severe human disease is unknown .	in total , we received six blackbirds , five owls , and one swallow for investigation . at necropsy ( estimated postmortem times between 24 h and 48 h ) , paraffin - embedded tissue samples were immunostained with a polyclonal mouse antibody to wnv ( b. murgue , institut pasteur , paris ) and a polyclonal rabbit antibody to tick - borne encephalitis virus ( [ tbev ] strain neudoerfl , h. holzmann , klinisches institut fr virologie , vienna ) using the avidin - biotin complex technique ( 8) . rna was extracted from 140-l organ homogenates or cell culture suspensions by using the qiaamp viral rna mini kit ( qiagen gmbh , hilden , germany ) . after aligning available nucleotide ( nt ) sequences of various mosquito - borne flaviviruses and determining highly conserved genomic regions , we designed three pairs of oligonucleotide primers ( to amplify a wide range of mosquito - borne flaviviruses ) and used them in the reverse transcription - polymerase chain reaction ( rt - pcr ) assays : 5-tacaacatgatgggvaaragagaga-3 ( nt position 90319055 of wnv genbank accession no . nc 001563 ) and 5-agcatgtcttcygtbgtcatccayt-3 ( nt position 1011510091 ) , resulting in a 1,084-bp amplification product ; 5-gartggatgacvacrgaagacatgct-3 ( nt position 1009010115 ) and 5-ggggtctcctctaacctctagtcctt-3 ( nt position 1083210807 ) , amplifying a 743-bp pcr product ; and 5-gccaccggaagttgagtaga-3 ( nt position 1046010479 of wnv no . nc 001563 ) and 5-gctggttgtgcagagcagaa-3 ( nt position 1090810889 ) , resulting in a 449-bp amplicon . reverse transcription and amplifications were performed in a continuous rt - pcr method by using the qiagen onestep rt - pcr kit ( qiagen gmbh ) . each 25-l reaction mixture contained 5 l 5x buffer ( final mgcl2 concentration 2.5 mm ) , 0.4 mm deoxynucleoside triphosphate ( dntp ) , 10 u recombinant rnasin ribonuclease inhibitor ( promega , madison , wi ) , 40 pmol forward and reverse primers , 1 l enzyme mix , and 2.5 l template rna . reverse transcription was performed for 30 min at 50c . following an initial denaturation for 15 min at 95c , the reaction mixture was subjected to 45 cycles of heat denaturation at 94c for 30 s , primer annealing at 60c for 30 s , and dna extension at 72c for 1 min , completed by a final extension of 10 min at 72c . following rt - pcr , we performed electrophoresis on 20 l of the amplicons in a 1.2% tris acetate - edta - agarose gel . the gel was stained with ethidium bromide , and the bands were observed under uv light . the pcr products were sequenced in both directions by using the abi prism 310 genetic analyzer automated sequencing system ( perkin elmer instruments , wellesley , ma ) . the nucleotide sequences were compiled and aligned with the corresponding sequences deposited in the genbank database . finally , we constructed a phylogenetic tree based on a 1,035-nt fragment in the ns5 genomic region . the following sequences have been included in the phylogenetic analysis : af013384 , koutango virus ; af013413 , yaounde virus ; d00246 , kunjin virus ; af202541 , wnv ( new york 1999 ) ; m12294 , wnv ; af013367 , cacipacore virus ; u15763 , jev ; af013360 , alfuy virus ; af013389 , mvev ; af013412 , usuv ( south africa ) ; af452643 , usuv ( austria ) ; af013416 , slev ; m93130 , denv ( type 3 ) ; and af013417 , yfv . the phylogenetic analysis was carried out by using phylogeny interference program package ( phylip ) , version 3.57c ( available from : url : http://evolution.genetics.washington.edu/phylip.html ) . distance matrices were generated with the dnadist / neighbor - joining program ; a translation / transversion ratio of 2.0 . paraffin - embedded tissue samples were processed as described ( 9 ) . for detection of wnv nucleic acid , an antisense digoxigenin - labeled riboprobe complementary to nt 49665439 of wnv strain ny1999 was generated from plasmid pwnny-88b-14 ( w.i . the final concentration of the probe was approximately 0.5 ng/l . for detection of usuv nucleic acid , we used a digoxigenin - labeled oligonucleotide probe with the sequence : 5-tcgcataactttcaccaccttgtgtttgtaggtcagctc-3 , which is complementary to nt 367 - 328 of the accessible partial sequence of the ns5 gene of usuv ( genbank accession no . necropsy showed grossly swollen livers and spleens , as well as seromucous enteritis in all blackbirds and owls ; histology showed various degrees of multifocal acute necrosis in liver and spleen . although the blackbirds did not show obvious histologic brain lesions , the owls had encephalitis , predominantly shown as multifocal areas of neuronophagia and microgliosis ( figure 1 , a and d ) . pathologic and immunohistochemical investigation of the swallow did not yield useful results because of severe autolysis . histology and detection of viral signals in paraffin - embedded tissue sections of birds infected with usutu virus ( usuv ) . f , great gray owl ; a , no histologic lesions present , hematoxylin and eosin staining ; b immunohistochemistry , using a polyclonal antibody to west nile virus , shows numerous positive neurons ; c , in situ hybridization with usuv - specific oligonucleotide probe shows a staining pattern comparable with that in b ; d , microglial nodule within the cerebral cortex , hematoxylin and eosin staining ; e , immunohistochemistry shows single positive neurons within a glial nodule ; f , in situ hybridization shows several positive neurons next to a glial nodule . original magnification , x 130 ( a c ) , x 200 ( d f ) . immunohistochemistry ( ihc ) with polyclonal antibodies to wnv was positive in 10 of 11 brains , showing reaction products in neurons and their processes and in the cytoplasm of microglial cells in glial nodules ( figure 1 , b and e ) . positive reactions were also present in kidney , spleen , liver , lung , and autonomous ganglia of the gastrointestinal tract . brain and kidney samples from wnv - infected birds from the united states and israel , respectively , were positive controls ; blackbirds that died from trauma were negative controls . ihc with polyclonal antibodies to tbev , another flavivirus found in central europe , showed negative results . rt - pcr with wnv - specific primers and ish with a wnv - specific probe were negative . infection with a flavivirus related to wnv would account for the cross - reactivity of a polyclonal antibody used in ihc and the negative outcome of the wnv - specific assays . after 2448 hours , a cytopathic effect of cell rounding could be observed ; 1 to 2 days later , the affected cells detached and floated in the medium . rt - pcrs with universal flavivirus primers resulted in clear pcr amplification products of the expected lengths . the primers were designed to amplify overlapping pcr products in the ns5 genomic region of mosquito - borne flaviviruses . rt - pcrs were performed both on the original organ homogenates and on cell culture suspensions , with identical results . despite a poor state of preservation , organ homogenates of the swallow the pcr products ( 1,084 bp , 743 bp , and 449 bp ) were directly sequenced in both directions ; the compiled nucleotide sequences ( a stretch of 1,877 bp , representing approximately 17% of flavivirus genome ) were aligned and compared with other sequences by using the basic local alignment search tool ( blast ) search ( national center for biotechnology information , national institutes of health , bethesda , md ) . the sequence obtained from the austrian dead birds was 97% identical with a 1,035-nt fragment of usuv ( genbank accession no . af013412 ; ) , an african mosquito - borne flavivirus in the jev group . to investigate the genetic relationship of the austrian usuv with other flaviviruses in the jev antigenic complex , we constructed a phylogenetic tree by using the programs in the phylip package ; we also included in the phylogenetic analysis three other important mosquito - borne flaviviruses ( yfv , slev , and denv3 ) . the phylogenetic tree ( figure 2 ) demonstrates the close genetic relationship between usuvs isolated in south africa and in austria ; therefore , we classified the austrian usuv as part of the jev group of flaviviruses . at the amino - acid level , the austrian and the south african usuv isolates proved ( in the investigated 1,035-nt region ) to be 100% identical . phylogenetic analysis of several members of the japanese encephalitis virus ( jev ) group and selected other mosquito - borne flaviviruses demonstrates the close genetic relationship of the austrian usutu virus ( usuv ) isolate with the south african usuv ( red underlay ) ; well - known members of the jev group are highlighted in red ; distinct branches are formed by saint louis encephalitis virus , dengue virus ( type 3 ) , and yellow fever virus . the partial nucleotide sequence of the austrian usuv isolate used in the phylogenetic tree has been deposited in the genbank database under accession no . af452643 . with an oligonucleotide probe specific for usuv , ish showed presence of viral nucleic acid in the cytoplasm of neurons in a distribution pattern closely matching ihc ( figure 1 , c and f ) . regarding other organs , however , kidneys of only two birds were positive , probably reflecting rna degradation due to postmortem times > 24 h. we demonstrated the presence of a mosquito - borne flavivirus , never before observed outside tropical and subtropical africa , in the continental climate of central europe , where winter temperatures are as low as 20c . since we also detected usuv nucleic acid in a barn swallow , the virus was probably introduced to the austrian bird population by swallows or other migrating birds . bird die - offs in various bird species in different areas of austria suggest that the virus has already adapted to local mosquito species , which are probably transmitting the virus . isolating the virus from local mosquitoes has not been attempted thus far but is planned . during a retrospective survey of paraffin - embedded blackbird tissues by ihc and rt - pcr , we also detected usuv in a blackbird that died a year earlier ( 2000 ) . a partial nucleotide sequence of this usuv proved to be 100% identical to the sequences of the 2001 usuv isolates . although no severe bird die - offs were observed in 2000 , we think that usuv may already be established and be overwintering in austria ( rather than being newly introduced in 2 consecutive years ) . comparable with the introduction of wnv to north america in 1999 , where the virus propagated in local mosquito species and then rapidly spread from new york to > 20 states in the united states and canada ( 10 ) , we foresee a similar scenario for usuv in europe . this study shows for the first time that usuv is highly pathogenic for several different species of birds . because full sequence data of usuv isolates are not available ( only a short [ 1,035-bp ] fragment of one usuv isolate has been deposited in the genbank database ) we can not provide information on amino acid changes that might contribute to altered pathogenic properties . for the closely related wnv , the pathogenicity for birds seems to depend on the virus strain and whether the virus affects a previously exposed or unexposed population . the wnv strain that appeared in north american birds in 1999 is closely related to a strain isolated in israel ; this strain is associated with avian deaths in both countries ( 1114 ) . certain other wnv strains , such as those responsible for recent outbreaks in romania and russia in humans ( 15,16 ) and in italy and france in equines ( 17,18 ) , were not associated with avian deaths . also , the fact that certain avian species , such as eurasian blackbirds , great gray owls , and barn swallows in austria , are especially vulnerable to usuv infection , is reminiscent of the observation that wnv in north america has primarily affected american crows and blue jays ( 19,20 ) . the emergence of wnv in the united states in 1999 and usuv in central europe in 20002001 is an indication of future virus activity . the next mosquito - borne flavivirus , which might be introduced to regions far from its original habitats , may be highly pathogenic for humans , farm animals , or pets , as many strains of jev group are . we could speculate whether global warming or other environmental factors may have contributed to the introduction and maintenance of usuv , formerly restricted to tropical and subtropical areas , in a much colder climate . as a consequence of the introduction of usuv to central europe , surveillance programs for mosquito - borne flaviviruses in general ( based on virus detection in mosquitoes and dead birds , as well as epidemiologic investigations ) should be established in europe , like those initiated in the united states after the first occurrence of wnv ( 19,20 ) . moreover , we will have to fully sequence usuv , establish serologic test systems , and evaluate the spread and pathogenic potential to control this new virus infection in central europe .
backgroundto treat moderate to severe acne vulgaris , we developed an optical fiber imported intra - tissue photodynamic therapy : the optical fiber irradiation 5-aminolevulinic acid photodynamic therapy ( ofi - ala - pdt ) . the aim of this study was to compare the treatment effect and tolerability of ofi - ala - pdt versus traditional ala - pdt in the treatment of moderate to severe acne vulgaris.material/methods60 patients with facial acne enrolled into this study were randomly divided into an ofi - ala - pdt group and a traditional ala - pdt group , with 30 patients in each group . the difference between these 2 groups was the red light irradiation methods used . in the ofi - ala - pdt group we used intra - tissue irradiation ( import the red light directly into the target lesion with optical fiber ) for 5 min , while the traditional ala - pdt group received whole - face irradiation for 20 min . all patients received 1 irradiation every 7 to 10 days for a total of 6 irradiations . treatment effects and adverse reactions were recorded after the 4th and 6th irradiation , and at 4 , 8 , 16 weeks after the entire treatment.resultsafter the 4th irradiation , significantly different effective rates were observed in these groups ( 90.0% for the ofi - ala - pdt group and 66.7% for the control group ) . however , no significant difference in effective rate was recorded in the later observations . there were 182 adverse reactions in the ofi - ala - pdt group and 497 in the control group , which showed a significant difference ( p<0.05).conclusionsofi - ala - pdt showed improved treatment effective rate in the early stage of irradiation , and it had fewer adverse reactions .	the recommended treatments for acne include topical medications and systemic use of antibiotics , hormones , and retinoids , according to severity . tretinoin or isotretinoin is very effective for the treatment of intractable acne but its use has been limited due to the potential for teratogenicity . topical photodynamic therapy ( pdt ) with topical application of sensitizers in the treatment of different kinds of skin diseases is of increasing research interest . pdt is based on the activation of light - sensitive molecules ( photosensitizers ) to cause cell death by producing cytotoxic oxygen radicals . 5-aminolevulinic acid ( ala ) is frequently used in topical pdt because it is converted into protoporphyrin ( ppix ) , an extremely active photosensitizer , upon irradiation with red light . recent studies have suggested that ala - pdt is also effective in treatment of acne vulgaris . with the increasing use of ala - pdt for patients with acne vulgaris , adverse effects have increased , including pain , burning sensation , itching , prickling , erythema , edema , pustules , acneiform eruption , and excoriations [ 57 ] , with causes related to the concentration of photosensitizer , the energy of light , and skin conditions of patients . therefore , we studied an optical fiber intra - tissue irradiation 5-aminolevulinic acid photodynamic therapy ( ofi - ala - pdt ) in the treatment of moderate to severe acne in patients treated in our department from january 2014 to june 2015 . patient inclusion criteria were according to the diagnosis and classification in the cunkuffe classification method . these classifications areas are : light ( mainly whiteheads and blackheads ) , medium ( mainly inflammatory papules and pustules ) , and severe ( inflammatory papules , nodules , and inflammatory cysts ) . exclusion criteria were : ( 1 ) internal or external use of antibiotics within the last 4 weeks ; ( 2 ) systemic use of a retinoid in the last 6 months ; ( 3 ) photosensitive or keloid history ; ( 4 ) pregnant or liver function unusual ; ( 5 ) can not complete the course ; ( 6 ) staff directly participating in the study ; and ( 7 ) participants currently in other clinical studies or who participated in another study within the last 3 months . the study was upon the approval of the hospital ethics committee and all the patients signed informed consent . these 60 patients with moderate to severe acne were treated from january 2014 to june 2015 in our dermatology department . rash was limited to the face , and the main symptoms were inflammatory papules , pustules , nodules , and cysts , with different degrees of pigmentation and scarring . the treatment group ( ofi - ala - pdt ) included 12 males and 18 females , aged 1244 years , with an average of 26.85.2 years ; the course of disease ranged from 3 to 120 months with an average of 30 months , with no drop - outs . in the control group , there were 16 males and 14 females , aged 1542 years with an average of 27.34.8 years ; the course of disease ranged from 4 to 108 months , with an average of 33 months , with no drop - outs . no significant differences were observed between the 2 groups in sex , age , or course of disease ( p>0.05 ) . in the treatment group ( ofi - ala - pdt ) ( figure 1 ) , 3.6% aminolevulinic acid was evenly applied to the rashes and the surrounding 0.5 to 1.0 cm of normal skin . after 1.5 h of incubation shielded from light , we wiped off the remaining photosensitizer , disinfected , inserted disposable optical fiber needles into the skin lesions with inflammatory papules and nodules , and imported the red light irradiation to the tissues located 3 mm below the follicular orifice ( including the sebaceous glands ) for 5 min . a dose of 4.5 j / cm ( dose at skin , detected by a vlp-200 laser power meter , changchun feimiao tech . , ltd ) was given for the first time ( this dose was based on our previous data in an animal model , data not shown ) , and was adjusted to 324 j / cm in the following irradiations according to adverse reactions . irradiations were carried out every 710 days , and each patient received 6 irradiations in total . in the control group , the traditional skin surface irradiation was used . for irradiation , a dose of 54 j / cm at skin was used with a fixed - power density of 45 mw / cm for 20 min , and the distance between the light panel and patient s apex nasi was set at 10 cm . effective criteria were the number of skin lesions and curative effect as recorded and judged by the same dermatologist . cure was 90% or more of the skin lesions disappeared ; remarkably effective was 60% to 89% of the skin lesions disappeared ; effective was 20% to 59% of skin lesions disappeared ; and invalid was less than 20% of skin lesions disappeared . effective rate was the percentage of cured cases plus remarkable cases divided by the total cases . treatment effects and adverse reactions were recorded during each treatment , before the next treatment and in the subsequent follow - up period . these adverse reactions include itching , pain , pustules , blisters , edematous erythema , pigmentation , reactive acne , and desquamation . we recorded the appearing and fading away time , severity , and actions used to combat these adverse reactions . follow - up was performed at 4 , 8 , and 16 weeks after the last irradiation . the chi - square test was used to compare different groups and the rank - sum test was used to compare adverse reactions between groups , age , and disease courses . patient inclusion criteria were according to the diagnosis and classification in the cunkuffe classification method . these classifications areas are : light ( mainly whiteheads and blackheads ) , medium ( mainly inflammatory papules and pustules ) , and severe ( inflammatory papules , nodules , and inflammatory cysts ) . exclusion criteria were : ( 1 ) internal or external use of antibiotics within the last 4 weeks ; ( 2 ) systemic use of a retinoid in the last 6 months ; ( 3 ) photosensitive or keloid history ; ( 4 ) pregnant or liver function unusual ; ( 5 ) can not complete the course ; ( 6 ) staff directly participating in the study ; and ( 7 ) participants currently in other clinical studies or who participated in another study within the last 3 months . the study was upon the approval of the hospital ethics committee and all the patients signed informed consent . these 60 patients with moderate to severe acne were treated from january 2014 to june 2015 in our dermatology department . rash was limited to the face , and the main symptoms were inflammatory papules , pustules , nodules , and cysts , with different degrees of pigmentation and scarring . the treatment group ( ofi - ala - pdt ) included 12 males and 18 females , aged 1244 years , with an average of 26.85.2 years ; the course of disease ranged from 3 to 120 months with an average of 30 months , with no drop - outs . in the control group , there were 16 males and 14 females , aged 1542 years with an average of 27.34.8 years ; the course of disease ranged from 4 to 108 months , with an average of 33 months , with no drop - outs . no significant differences were observed between the 2 groups in sex , age , or course of disease ( p>0.05 ) . in the treatment group ( ofi - ala - pdt ) ( figure 1 ) , 3.6% aminolevulinic acid was evenly applied to the rashes and the surrounding 0.5 to 1.0 cm of normal skin . after 1.5 h of incubation shielded from light , we wiped off the remaining photosensitizer , disinfected , inserted disposable optical fiber needles into the skin lesions with inflammatory papules and nodules , and imported the red light irradiation to the tissues located 3 mm below the follicular orifice ( including the sebaceous glands ) for 5 min . a dose of 4.5 j / cm ( dose at skin , detected by a vlp-200 laser power meter , changchun feimiao tech . , ltd ) was given for the first time ( this dose was based on our previous data in an animal model , data not shown ) , and was adjusted to 324 j / cm in the following irradiations according to adverse reactions . irradiations were carried out every 710 days , and each patient received 6 irradiations in total . in the control group , the traditional skin surface irradiation was used . for irradiation , a dose of 54 j / cm at skin was used with a fixed - power density of 45 mw / cm for 20 min , and the distance between the light panel and patient s apex nasi was set at 10 cm . effective criteria were the number of skin lesions and curative effect as recorded and judged by the same dermatologist . cure was 90% or more of the skin lesions disappeared ; remarkably effective was 60% to 89% of the skin lesions disappeared ; effective was 20% to 59% of skin lesions disappeared ; and invalid was less than 20% of skin lesions disappeared . effective rate was the percentage of cured cases plus remarkable cases divided by the total cases . treatment effects and adverse reactions were recorded during each treatment , before the next treatment and in the subsequent follow - up period . these adverse reactions include itching , pain , pustules , blisters , edematous erythema , pigmentation , reactive acne , and desquamation . we recorded the appearing and fading away time , severity , and actions used to combat these adverse reactions . follow - up was performed at 4 , 8 , and 16 weeks after the last irradiation . the chi - square test was used to compare different groups and the rank - sum test was used to compare adverse reactions between groups , age , and disease courses . the treatment results are shown in figure 2 . by the end of the 4 irradiation , the effective rate of the ofi - ala - pdt group was 90% , which is significantly higher than the control group s 66.7% ( =4.812 , p<0.05 ) . however , there was no significant difference between these groups when all the 6 irradiations were finished ( 93.3% in the treatment versus 90% in the controls ) ( table 1 ) . furthermore , follow - up at 4 , 8 , and 16 weeks after all the treatment showed no improvement in the ofi - ala - pdt group ( table 1 ) . there were 84 adverse reactions recorded in the ofi - ala - pdt group during treatment , which is significantly less than the 242 recorded in the control group ( table 2 ) . after the treatment , there are 98 adverse reactions in the treatment group and 255 in the control group ( table 2 ) . overall , 182 adverse reactions in the ofi - ala - pdt group and 497 in the control group were recorded , indicating that ofi - ala - pdt treatment performed better in decreasing the number of adverse reactions ( p<0.01 ) . the treatment results are shown in figure 2 . by the end of the 4 irradiation , the effective rate of the ofi - ala - pdt group was 90% , which is significantly higher than the control group s 66.7% ( =4.812 , p<0.05 ) . however , there was no significant difference between these groups when all the 6 irradiations were finished ( 93.3% in the treatment versus 90% in the controls ) ( table 1 ) . furthermore , follow - up at 4 , 8 , and 16 weeks after all the treatment showed no improvement in the ofi - ala - pdt group ( table 1 ) . there were 84 adverse reactions recorded in the ofi - ala - pdt group during treatment , which is significantly less than the 242 recorded in the control group ( table 2 ) . after the treatment , there are 98 adverse reactions in the treatment group and 255 in the control group ( table 2 ) . overall , 182 adverse reactions in the ofi - ala - pdt group and 497 in the control group were recorded , indicating that ofi - ala - pdt treatment performed better in decreasing the number of adverse reactions ( p<0.01 ) . the clinical treatment for moderate to severe acne has mainly focussed on the systematic usage of antibiotics and retinoids . however , this kind of treatment is limited due its obvious adverse effects . in recent years , a large number of studies have confirmed that ala - pdt by red light is a highly effective treatment for acne [ 1014 ] . unfortunately , with the extensive use of ala - pdt , more and more adverse effects had been reported . almost all photodynamic therapy patients experience various ranges of adverse reactions , including moderate to severe pain , erythema , pustules , and peeling . the presently known factors affecting photodynamic therapy efficiency and safety include the concentration of photosensitizer , photosensitizer packet time , photosensitizer type , light type , and light parameters . the conventional method used to reduce adverse reactions is to avoid light and sunshine , and cold spray or cold repair facial mask after photodynamic therapy , but the effect is limited . one study used different concentrations of ala for the treatment and found that the higher concentration was related to improved curative effect but caused severe adverse reactions . some studies reduced the ala packet time from a few hours to 1 hour and observed low incidence of adverse reactions , especially in the case of pigmentation , but its curative effect is reduced to a certain degree . at the same time , the use of new photosensitizers is also a popular research topic . jang introduced new photosensitizers ( indocyanine green and indole-3-acetic acid ) in photodynamic therapy for acne . the study achieved a good effect , and only produced slight erythema ( 6/34 ) and pruritus ( 8/34 ) after each irradiation , which resolved within a few hours . the photodynamic approach currently used in the ala - pdt uses the external exposure mode . in this mode , to get to the hair follicle sebaceous glands , the red light has to penetrate the skin epidermis layer . however , this is bound to increase the light attenuation , and the epidermis layer may absorb more light , leading to local adverse effects . on the other hand , the hair follicle sebaceous glands would absorb less energy and the effect would be limited . therefore , to address this problem , we used disposable optical fiber needles to penetrate the skin 3 mm to import the red light into subcutaneous layers of the target site . this method was expected to achieve accurate , minimally invasive , targeted therapy in the tissues and reduce damage to the normal skin surrounding the lesions , so it can alleviate the related adverse effects . to determine whether this irradiation method could improve the therapeutic effects , as expected , by the end of the 4 irradiation , the effective rate of the ofi - ala - pdt group reached 90% , which is significantly higher than the 66.7% achieved in the control group ( =4.812 , p<0.05 ) . these results indicate the ofi - ala - pdt contributed to the effective rate in the early stage of irradiation but did not help the long - time therapy . moreover , 182 adverse reactions in the ofi - ala - pdt group and 497 in the control group were recorded , indicating that ofi - ala - pdt treatment performed better in decreasing the adverse reactions ( p<0.01 ) . overall , the ofi - ala - pdt treatment developed in this study showed improved treatment effective rate in the early stage of irradiation and fewer adverse reactions .
the catecholamine biosynthetic pathway consists of several enzymatic steps in series , beginning with the amino acids phenylalanine and tyrosine , and eventuating in the catecholamines norepinephrine ( noradrenaline ) and epinephrine ( adrenaline ) . since the enzyme tyrosine hydroxylase ( th ; tyrosine 3-mono - oxygenase ; ec 1.14.16.2 ; chromosome 11p15.5 ) is generally considered to be rate - limiting in this pathway , probed as to whether common genetic variation at the th gene occurred , and whether such variants contributed to inter - individual alterations in autonomic function , either biochemical or physiological . we began with sequencing a tetranucleotide ( tcat ) repeat in the first intron , and found that the two most common versions , ( tcat)6 and ( tcat)10i , predicted heritable autonomic traits in twin pairs . we then conducted systematic polymorphism discovery across the ~8 kbp locus , and discovered numerous variants , principally non - coding . the proximal promoter block contained four common variants , and its haplotypes and snps ( especially c-824 t , rs10770141 ) predicted catecholamine secretion , environmental stress - induced bp increments , and hypertension . finally , we found that two of the common promoter variants , c-824 t ( rs10770141 ) and a-581 g ( rs10770140 ) , were functional in that they differentially affected transcriptional activity of the isolated promoter , disrupted recognition motifs for specific transcription factor binding , altered the promoter responses to the co - transfected ( exogenous ) factors , and bound the endogenous factors in the chromatin fraction of the nucleus . we concluded that common variation in the proximal th promoter is functional , giving rise to changes in autonomic function and consequently cardiovascular risk .	tyrosine hydroxylase ( th ; tyrosine 3-mono - oxygenase ; ec 1.14.16.2 ; chromosome 11p15.5 ) , which catalyzes the formation of dopa from dopamine , is generally considered to be the rate - limiting enzyme in the catecholamine biosynthetic pathway ( flatmark and stevens 1999 ) . although a single - copy gene , th produces four different types of mrna through alternative splicing of a single primary transcript ( grima et al . substantial loss of th enzymatic activity has profound consequences both in humans ( flatmark and stevens 1999 ) and in mice with targeted ablation of the th locus ( zhou et al . the human th locus bears several examples of common natural allelic variation , such as the tetranucleotide repeat [ or microsatellite polymorphism , ( tcat)n ] in its first intron , which associate with essential hypertension ( sharma et al . , 1998 ) . the th tetranucleotide repeat ( tcat)n occurs in 511 head - to - tail copies . ( 2003 ) found that ( tcat)6 and ( tcat)10i alleles seemed to be cardioprotective by association with attenuation of the hemodynamic response to stress with increasing age , while ( tcat)7 seemed to be deleterious by their association with higher resting systolic blood pressure and greater hemodynamic response to stress with increasing body mass index . sharma et al . ( 1998 ) found that the ( tcat)10 ( e ) allele was over - represented in hypertensive subjects , whereas the ( tcat)9 ( d ) allele frequency was increased in control ( normotensive ) subjects . wei et al . ( 1997 ) found ( tcat)9 to be associated with higher norepinephrine levels . in order to probe the role of th polymorphism in stress - induced disease pathways 2002 ) . exploring the effects of particular ( tcat)n alleles on intermediate phenotypes , we stratified each individual on the basis of number of copies of the allele . we found that the two most common alleles , ( tcat)6 and ( tcat)10i , influenced a number of autonomic traits , both biochemical and physiological : ( tcat)6 copy number affected basal pulse interval and heart rate , and post - stress heart rate . ( tcat)10i copy number affected basal pulse interval , plasma epinephrine , and renal norepinephrine excretion ( zhang et al . we documented the heritability of blood pressure , and found that ( tcat)6 allele frequencies differed among individuals stratified by genetic risk ( family history ) of hypertension : in particular , family history - positive individuals were less likely to bear the ( tcat)6 allele . since increasing ( tcat)6 allele copy number was associated with lower basal and stress - induced heart rates , the results obtained suggested a mechanism whereby ( tcat)6 alleles may be protective against the future development of hypertension . the ( tcat)n motif can bind such transcription factors as ap1 or znf191 ( albanese et al . 2001 ) and may function as a transcriptional enhancer when tested in transfected / expressed promoter / reporter plasmids . however , in transfected th promoter / intron / reporter studies ( albanese et al . 2001 ) , the ( tcat)n repeat silences transcription in a copy number - dependent way ; by contrast , in vivo we observed directionally opposite associations of common ( tcat)n alleles with autonomic function : ( tcat)10i with activation and ( tcat)6 with diminution of sympathetic outflow ( zhang et al . ( tcat)n could not mechanistically explain common variation in human autonomic function . therefore , we carried out systematic polymorphism discovery at the locus . in order to probe the possible underlying impact of th variation on stress - induced disease pathways , we re - sequenced ~1.2 kbp of 5 promoter as well as all 13 exons and adjacent intronic regions for rare and common variants in 80 ethnically diverse subjects as well as 422 twins . plasma and urine catecholamines were measured by radioenzymatic assay ( kennedy and ziegler 1990 ) . in the coding region , we found two common bi - allelic variants , only one of which was non - synonymous : val81met ( at 37.4% ) which did not associate with the autonomic traits we studied . we also found 12 unusual coding region variants , but the non - synonymous variants had minor allele frequencies of only 0.30.6% , not sufficient to account for the common population associations . thus , we turned to potential regulatory ( non - coding ) variants and found 10 snps in the proximal promoter region , four of which ( c-824 t , g-801c , a-581 g , and g-494a ) were common ( minor allele frequencies > 10% ) . statistically , c-824 t ( rs10770141 ) and a-581 g ( rs10770140 ) seemed to influence both catecholamine secretion and the blood pressure response to environmental stress . a multivariable analysis further indicated that c-824 t became the most significant predictor of change in dbp during cold stress . the -824 t allele is associated with increased catecholamine production , increased blood pressure increments in response to stress , and extreme blood pressure values in the population . it was tempting to speculate that the functional variation that we observed in -824 t carriers could be the outcome of environmental selective pressures on alleles augmenting catecholaminergic function ( rao et al . more recently , we performed additional studies to test whether such th promoter variants were by themselves functional ( zhang et al . first , common haplotypes ( at least 15% frequency ) of the th promoter were generated by site - directed mutagenesis , then verified by sequencing , and inserted into luciferase reporter vectors . after transfection into pc12 cells , such human th promoter haplotypes showed substantial differences in luciferase reporter activity . by two - way anova on luciferase activity , two of the four common variants ( c-824 t [ rs10770141 ] and a-581 g [ rs10770140 ] ) altered transcriptional activity . comparison of these in cells results with the effects of the same 4 variants in vivo indicates that the same variants ( c-824 t and a-581 g ) that exert the greatest effect on th transcription in cells also have the most pronounced effect on human catecholamine secretion in vivo . in addition , the -824 t allele displayed an augmented response to typical chromaffin cell secretory stimuli , such as nicotine or pacap . under basal circumstances , greater activity of the t allele was observed , and when stimulated by nicotine , c-824 t responded differentially , with an increased effect of the drug on the t allele ; when stimulated by pacap , the increased response of the t allele was even more apparent . the -581 g allele also responded differentially to pacap . in the basal state , greater activity of the g allele was apparent ; during nicotine stimulation , greater activity of the g allele was noted , as was the case during response to pacap . we further probed the functional significance of c-824 t and a-581 g by co - transfection of sequence - predicted trans - activating factors in cells : mef2 , sry , and foxd1 differentially activated c-824 t , whereas the g / c - rich binding factors sp1 , ap2 , and egr1 differentially activated a-581 g . at c-824 t , the co - transfected human mef2 increased th promoter expression to 131.3% on the t allele , but had little effect on the c allele . the mef2 dominant - negative mutant mmef2 decreased th promoter expression down to 71.3% on the t allele , but had little effect on the c allele . thus , at c-824 t , factor mef2 acted in a directionally coordinate fashion ( at t > c ) to explain the in vivo trait associations , whereas at a-581 g , factors sp1 , ap2 , and egr1 displayed similar differential actions ( at g > a ) . chromatin immunoprecipitation ( chip ) confirmed the interaction of the endogenous transcription factors with these motifs in the nucleus ( zhang et al . we conclude that inter - individual variability in catecholamine secretion is controlled in substantial part by genetic variation in the adrenergic pathway encoding catecholamine synthesis , especially at the classical rate - limiting step , th . c-824 t and a-581 g are not only statistical predictors of catecholamine secretory and stress bp response traits in vivo , but also causally responsible for alterations in transcriptional efficiency of the th gene . our results thus document novel pathophysiological links between a key adrenergic locus , catecholamine metabolism , and blood pressure , and suggest new strategies to approach the mechanism , diagnosis , and treatment of autonomic dysfunction as well as systemic hypertension .
the aluminum concentration and ryznar index increased and the ph decreased in a small appalachian water supply reservoir following acid precipitation runoff episodes . concomitant increases in tapwater aluminum and decreases in tapwater ph were also observed at two homes in the water distribution system . lead concentrations in the tapwater of one home frequently exceeded recommended levels , although spatial and temporal variation in tapwater copper and lead concentrations was considerable . since source water and reservoir water copper and lead concentrations were much lower , the increased copper and lead concentrations in tapwater were attributed to corrosion of household plumbing . tapwater copper concentration correlated well with tapwater ph and tapwater temperature . asbestos fibers were not detected in tapwater . the asbestos - cement pipe in the water distribution system was protected by a spontaneous metallic coating that inhibited fiber release from the pipe . several simultaneous reactions were hypothesized to be taking place in the distribution system that involved corrosion of metallic components and coating of asbestos - cement pipe components in part with corrosion products and in part by cations of watershed origin . greater water quality changes might be expected in areas of higher atmospheric deposition.imagesfigure 5.figure 6 .
setting . a nationwide study in fiji . objective . to describe the incidence of congenital rubella syndrome ( crs ) and its relationship to the incidence of notified cases of rubella in fiji from 1995 to 2010 . design . descriptive , retrospective review of all recorded congenital abnormalities associated with live births in fiji over 16 years . results . there were 294 infants who met the criteria for crs . of these , 95% were classified as suspected cases , 5% were clinically confirmed , and none were laboratory confirmed cases . there was a significant linear increase over the study period in the incidence of crs ( odds ratio 1.045 per year , 95% ci 1.019 to 1.071 , p 0.001 ) . there was no significant association between the incidence of crs and the reported incidence of rubella ( p = 0.3 ) . conclusion . there is a rising trend in reports of suspected crs cases in fiji . this highlights the need to strengthen surveillance for crs through improvements in clinical and laboratory diagnosis to confirm or exclude suspected cases . it is also important to ensure high coverage of rubella vaccination in fiji .	whilst rubella is usually a mild disease in adults and children , maternal infection with rubella , especially early in pregnancy , can cause severe defects in the developing foetus , resulting in congenital rubella syndrome ( crs ) . the constellation of anomalies of crs includes ophthalmic , auditory , cardiac , and craniofacial defects . crs is common in developing countries , affecting about 110,000 infants annually in these countries . in 2009 in addition , 165 cases of crs were reported to who by 123 member countries in the same year . in the western pacific region , the number of rubella cases increased 12-fold from 5475 in 2000 to 73077 in 2009 . with rubella a growing problem in the western pacific region , there are concerns that crs may also be on the rise in this region . the relationship between the incidence of rubella and the incidence of crs has not been clearly shown , although some studies from resource poor settings , such as romania , have shown clusters of children with crs after rubella outbreaks . in fiji , the incidence of rubella has ranged between 1 and 30 cases per 100,000 population with outbreaks noted in 1995 , 2002 , 2006 , and 2011 . the most recent rubella outbreak , in july 2011 , has highlighted the need to carry out surveillance of crs in fiji . vaccination against rubella was introduced in 1975 for females ( at 12 years of age ) , and in 2004 this was extended to include both males and females at primary school entry . however , a lack of information on the incidence of crs in fiji limits our ability to assess the effectiveness of these vaccination campaigns . to address this information gap , this study seeks to ( a ) report on the incidence of crs in fiji and the relationship between the incidence of crs and the incidence of notified cases of rubella since 1995 and ( b ) document how crs cases are classified ( suspected ( possible ) , clinically confirmed ( probable ) , or laboratory confirmed ( definite ) ) over the period 19952010 . this was a descriptive study involving a retrospective review of all recorded congenital abnormalities associated with live births in fiji over a 16-year period ( 19952010 ) . fiji is an island nation located in the south - west pacific with a population of approximately 837,271 . it consists of approximately 332 islands covering a total land area of about 18,333 sqkm . the ministry of health ( moh ) in fiji provides decentralized health services through a three tier structure of primary , secondary , and tertiary care . fiji 's health system comprises three divisional hospitals , 17 subdivisional hospitals , 78 health centres , and 103 nursing stations . there is a hierarchical referral mechanism from nursing stations to health centres to subdivisional and divisional hospitals . infants born with congenital defects requiring intervention are likely to be referred to the paediatric departments in the three divisional hospitals colonial war memorial ( cwm ) hospital , lautoka hospital , and labasa hospital . infants with newly diagnosed congenital anomalies are routinely admitted to intensive care wards at these hospitals . the study population included all live births in fiji with a congenital anomaly registered between january 1 , 1995 , and december 31 , 2010 . between november 2011 and april 2012 all newborn infants with congenital anomalies were identified from the congenital anomalies registers when these registers were available , and when not available , from a review of the neonatal intensive care unit registers . data on the number of live births and the annual population numbers were obtained from moh consolidated monthly reports ( cmr ) for the period 19952008 and from the moh public health information system ( phis ) for the period 2009 - 2010 . data on the annual number of reported cases of rubella were obtained from the national notifiable disease surveillance system reports for the period 19952010 . the cmr and phis records were used to allow calculation of the incidence rates of crs and rubella . for each recorded case of crs the following data were collected : hospital , date registered , date of birth , sex , ethnicity , and description of congenital defects . the presence or absence of specific congenital defects was recorded , as specified in the who diagnostic classification for crs and cases were classified as suspected , clinically confirmed , or laboratory confirmed where these notes were available , they were reviewed to verify the diagnosis that had been recorded in the congenital anomalies book or the neonatal intensive care unit register . the incidence of crs was expressed per 1000 live births , and the incidence of rubella was expressed as cases per 100,000 population for each year . the association between the incidence of rubella and the incidence of crs was estimated by linear regression using epi - info , version 3.5.1 . the congenital anomalies book was not available for lautoka hospital and for labasa hospital before 2010 . data was also missing for cwm hospital for 1998 and june december for 2000 . based on the available data , a total of 977 babies with congenital anomalies were recorded between 1995 and 2010 in fiji and initially 294 of these cases were found to meet the criteria for crs . folders were only available for 38 ( 13% ) of these 294 cases . in 33 cases the diagnosis was not changed but the classification was changed from suspected to clinically confirmed crs . in two cases crs cases comprised more males than females and i - taukei was the most common ethnic group followed by fijians of indian ethnic descent ( table 1 ) . between 1995 and 2010 , there was a significant linear increase in the incidence of crs ( odds ratio 1.045 per year , 95% ci 1.019 to 1.071 , p < 0.001 ) , with this incidence ranging from 0.4 cases per 1000 live births in 1995 to about 1.7 cases per 1000 live births in 2010 ( figure 1 ) . whilst the peak in incidence of rubella and the incidence of crs coincided in 2002 , there was no significant association between incidence of crs and rubella ( p = 0.3 ) ( figure 1 ) . the majority of crs cases ( n = 278 , 95% ) were classified as suspected thereafter , the annual proportion of clinically confirmed cases was consistently less than 20% of total crs cases . the most common presentations in babies with crs were congenital heart disease ( 80% ) followed by jaundice ( 10% ) . this study demonstrates an increasing trend in the incidence of suspected crs over the past 16 years . however , very few cases were clinically confirmed and there were no laboratory confirmed cases of crs . there was no significant relationship between the incidence of crs and the incidence of rubella . this study is based on data for crs recorded in all four divisions of fiji . we have ensured that the data are comparable among the hospitals and with international reports by using a standard criteria , as recommended by who , to classify the cases . there was a lack of information on clinical and laboratory aspects of the diagnosis in the registers . hence , we were not able to adequately assess the validity of the registers as a source of information on the diagnosis of crs . furthermore , not all hospitals had a congenital anomalies book and , where this did not exist , we could only obtain the diagnostic information by searching the neonatal intensive care admission book . the information contained in this book was also limited as it did not include laboratory results or detailed clinical findings . the range in incidence of possible crs recorded in fiji is similar to that reported in developing countries during epidemics at about 0.62.2 per 1000 live births [ 7 , 8 ] . this range of incidence is also similar to that of industrialized countries prior to vaccination [ 7 , 8 ] . whilst there was no significant relationship found between the incidence of rubella and incidence of crs in fiji , rubella outbreaks leading to increased incidences of crs have been documented in other countries such as panama , oman , and sri lanka . the majority of the crs cases are suspected cases in which the clinical finding was congenital heart disease . improvements in diagnosis of congenital heart disease over time could be contributing to increasing numbers of cases of suspected crs reported during this period . the low numbers of clinically confirmed cases reported may be due to difficulties in assessment at birth particularly for features that become more apparent at a later stage of life , for example , deafness , blindness , and mental retardation . the lack of a significant relationship between the incidence of crs and the incidence of rubella may be caused by underreporting of rubella due to shortcomings with data recording and management at the hospitals . the increasing incidence of suspected crs in fiji demonstrated through this study has implications for both clinical management and surveillance purposes . the absence of any laboratory confirmed crs cases suggests the need to develop standard operating procedures and guidelines for confirming diagnosis through serological tests so that we are able to accurately estimate the burden of crs in fiji . considering the challenges in documenting the extent of crs in the population due to great variation in the manifestations of crs in the first year of life and diagnostic issues related to detecting some of these features , a system would need to be established for close followup of these infants in maternal and child health clinics in order to identify features of crs that may not be possible to assess at birth . this study also suggests the need to monitor adequacy of rubella prevention , through the current rubella vaccination programme carried out at primary school entry . who recommends coverage rates to be maintained above 80% to ensure herd immunity . low coverage rates may indicate the need for serological surveys among women of child bearing age . furthermore , assessment of the sensitivity , specificity , and predictive value of the different clinical definition of crs may need to be carried out . the very small proportion of patient folders obtained for validation through this study suggests the need to improve information management and record keeping at health facilities . ultimately an improvement in surveillance of crs would allow evaluation of our disease control efforts towards elimination of rubella and crs . this highlights the need to strengthen surveillance for crs through improvements in clinical and laboratory diagnosis to confirm or exclude suspected cases .
objectivethe pseudomalabsorption of thyroxine has been used to describe patients with hypothyroidism who fail to comply with their treatment . we describe a unique case of a 32-year - old with hypothyroidism who developed pituitary hyperplasia and hyperprolactinemia secondary to the pseudomalabsorption of thyroxine.investigations and treatmentafter baseline thyroid - function tests were performed , the patient was administered levothyroxine 0.5 mg under the supervision of a registered nurse . thyroid function testing was repeated at 30 , 60 , 120 , and 180 minutes . arrangements were made for further daily supervised loading of levothyroxine 0.1 mg.resultswith the administration of 0.5 mg levothyroxine , free thyroxine levels increased by 120 minutes , and with daily supervised dosing of 0.1 mg there was normalization of the thyroid hormone levels and a reduction of thyroid - stimulating hormone levels . maintenance of thyroid - stimulating hormone < 15 mu / l for 2 weeks led to a reduction in prolactin levels and regression in the size of the pituitary on magnetic resonance imaging.conclusionif left untreated , these patients face significant morbidity and are at risk of developing pituitary hyperplasia , complications from an increase in pituitary size , hyperprolactinemia , and potentially myxedema coma . recognizing pituitary hyperplasia and hyperprolactinemia as a complication from the pseudomalabsorption of levothyroxine may prevent the potential of a misdiagnosis of a prolactinoma leading to unnecessary investigations and inappropriate treatment . patient awareness of this serious complication and the rapid , demonstrable resolution with adequate thyroid hormone replacement may provide motivation to comply with supervised dosing of levothyroxine . it has also been suggested that supervised treatment enables the individual to maintain their patient status , which may be in part the motivation behind this disorder .	primary hypothyroidism is a common medical condition , and may be a significant cause of morbidity if left untreated . the symptoms of hypothyroidism , including weight gain , constipation , fatigue , cold intolerance , hair loss , and poor concentration , can be debilitating . typically , the symptoms associated with this disorder are effectively treated with an oral replacement of thyroxine at an average daily dose of 1.6 mcg / kg ideal body weight.1 the correct dosing is usually confirmed with the resolution of symptoms and normalization of thyroid - stimulating hormone ( tsh ) levels within 68 weeks . one would expect that the debilitating nature of this disorder and the improvement with treatment would be reason enough for patients to comply with therapy . however , there have been a number of cases described where patients intentionally fail to take their prescribed thyroid medication.27 these patients are believed to suffer from an underlying psychiatric disorder ; more specifically , they are believed to suffer from factitious disorder , or as it has been termed by the authors in these cases , pseudomalabsorption of thyroxine . we describe a case report of a patient with a persistently elevated tsh level despite high doses of oral thyroxine who subsequently developed an enlarged pituitary and hyperprolactinemia . this article will highlight the factors that may interfere with thyroxine treatment , the consequences of untreated hypothyroidism and management of patients with pseudomalabsorption of levothyroxine . a 32-year - old woman presented with a 2-year history of elevated tsh levels ( > 100 mu / l , normal range 0.325.00 mu / l ) and undetectable free thyroxine / triiodothyronine ( ft4/ft3 ) levels despite increasing doses of oral thyroxine . at the time of presentation , she had been prescribed a thyroxine dose of 0.5 mg / day and liothyronine 0.125 mg daily . she had been on these doses for more than 6 months . on presentation , the patient complained of vague symptoms of weakness , but clinically there was no evidence of profound hypothyroidism . the patient was subsequently found to have an elevated prolactin level and enlarged pituitary on magnetic resonance imaging . table 1 provides details of the investigations and results that were carried out to rule out various factors that may have interfered with the absorption and clearance of thyroxine in this patient . with no evidence of malabsorption and absence of another medical condition to explain the lack of response to the high doses of levothyroxine , arrangements were made for supervised treatment with 0.5 mg of thyroxine treatment initially and then daily loading with 0.1 mg of levothyroxine and liothyronine 25 mcg for 5 days ( table 2 ) . a 32-year - old woman presented with a 2-year history of elevated tsh levels ( > 100 mu / l , normal range 0.325.00 mu / l ) and undetectable free thyroxine / triiodothyronine ( ft4/ft3 ) levels despite increasing doses of oral thyroxine . at the time of presentation , she had been prescribed a thyroxine dose of 0.5 mg / day and liothyronine 0.125 mg daily . she had been on these doses for more than 6 months . on presentation , the patient complained of vague symptoms of weakness , but clinically there was no evidence of profound hypothyroidism . the patient was subsequently found to have an elevated prolactin level and enlarged pituitary on magnetic resonance imaging . table 1 provides details of the investigations and results that were carried out to rule out various factors that may have interfered with the absorption and clearance of thyroxine in this patient . with no evidence of malabsorption and absence of another medical condition to explain the lack of response to the high doses of levothyroxine , arrangements were made for supervised treatment with 0.5 mg of thyroxine treatment initially and then daily loading with 0.1 mg of levothyroxine and liothyronine 25 mcg for 5 days ( table 2 ) . within 5 days of treatment , the patient s thyroid hormone levels normalized and tsh levels decreased to 7 mu / l . with tsh levels maintained at < 15 mu / l for 2 weeks , there was a reduction in the prolactin level and normalization of the pituitary on magnetic resonance imaging ( table 2 , figure 1 ) . although poor compliance with medication is the most common cause for treatment failure in patients with hypothyroidism,8 there are a number of pharmacological agents and disease processes that are known to affect the absorption or metabolism of levothyroxine ( table 3 ) . a number of studies have shown that both iron and calcium supplements can potentially decrease the absorption of thyroxine through the formation of a nonabsorbable complex in the gut.9,10 given the increased incidence of hypothyroidism in the elderly population and the association of both osteoporosis and anemia with this condition , it is important to consider the interaction of these supplements with thyroxine as a potential cause for treatment failure . to eliminate this interaction and to maximize the benefit of each treatment , other studies have shown that gastrointestinal disorders and anatomical gastrointestinal abnormalities , such as untreated celiac disease and short - gut syndrome , may affect the absorption of thyroxine.11,12 celiac disease is an important consideration in this group of patients , considering the autoimmune association between these two disorders and the frequency in which they occur in patients simultaneously . in terms of patients with celiac disease , normalization of thyroid hormone levels and tsh have been observed once a gluten - free diet has been implemented.11 normal absorption of thyroxine is also known to be affected by changes in the acidic environment of the stomach . specifically , it has been shown that patients with impaired gastric acid secretion , whether secondary to helicobacter pylori infection or atrophic gastritis , required higher doses of thyroxine - replacement therapy to become euthyroid.13 similar but reversible findings were also seen in patients treated with omeprazole , a proton - pump inhibitor used to suppress acid secretion.13 other common medications to consider in this group of patients are estrogen - replacement therapy and the selective estrogen receptor modulator raloxifene . arafah showed that a group of 18 postmenopausal women treated with thyroxine - replacement therapy demonstrated increases in serum thyroxine and thyroxine - binding globulin ( tbg ) concentrations while receiving estrogen treatment.14 however , patients with a diagnosis of hypothyroidism had decreased ft4 levels ( 22 5 pmol / l to 18 4 pmol / l ) and increased serum thyrotropin levels ( 0.9 1.1 to 3.2 3.1 ) . the coadministration of raloxifene and levothyroxine has also resulted in increased dosing requirements for thyroxine.15 it is not clear whether the increased dosing of thyroid - replacement treatment is related to an absorption problem or related to a change in the tbg level . garwood et al report a case where separation of these medications by 12 hours enabled their patient to reduce her thyroxine dosage , suggesting that it may be related to absorption , although the mechanism is unclear.15 in younger female patients , pregnancy should also be considered as a potential cause for increasing tsh levels and the need for an increase in thyroxine dosing , particularly in previously stable patients . similar to patients on estrogen - replacement therapy , the increased thyroid replacement dosing is attributed to an increase in tbg , resulting in decreased levels of ft4 and ft3 and subsequent rises in tsh levels.16 the increased thyroxine dosage requirements have been estimated at approximately 45% higher than their regular dose of thyroxine.16 once each of the various factors discussed above has been thoroughly investigated and eliminated , pseudomalabsorption of thyroxine should be considered in patients failing to achieve normal tsh despite high levels of thyroxine . this diagnosis and normal intestinal absorption can be confirmed through a supervised loading doses of up to 1 mg of oral thyroxine.5 it is often recommended that this be completed when patients are fasting , as uptake of thyroxine has been found to be greater in a fasting state.2 patients in these situations should respond with a rapid lowering of their tsh levels and an increase in ft4 levels . it has been reported that most levothyroxine absorption takes place within the first few hours of administration and that serum ft4 levels peak within 2 hours of administration.26 patients such as the one described in this case report are often emphatic about their compliance with the prescribed treatment . the obvious consequence of untreated hypothyroidism is the persistence of the common symptoms of fatigue , weight gain , constipation , and poor concentration , and the morbidity associated with each of these . however , there are more serious and potentially life - threatening consequences of hypothyroidism if left untreated . specifically , myxedema coma is a rare but serious medical emergency and definitely the most severe complication of decompensated hypothyroidism . it is often the result of coexisting medical conditions or drugs , and has been found to have a mortality rate of approximately 80% in untreated patients.1 another rare and potentially severe complication of untreated hypothyroidism is the development of a pituitary hyperplasia and hyperprolactinemia , as was seen in our patient . although there have been no documented cases where specifically the intentional noncompliance with thyroxine treatment has led to the development of pituitary hyperplasia , it is well established that one of the potential causes for hyperprolactinemia and pituitary hyperplasia is hypothyroidism.26,27 in these situations , the increase in pituitary size and elevated prolactin secretion are felt to be secondary to increased synthesis of thyroid releasing hormone ( trh ) in response to chronically low thyroid hormone levels . the development of pituitary hyperplasia in itself has the potential for quite severe complications , including headaches , neurological deficits , and visual loss . prolonged hyperprolactinemia may lead to infertility , hypopituitarism , and low bone density.28 although hypothyroidism is clearly not the only cause of a pituitary hyperplasia and elevated prolactin levels , resolution of these findings with appropriate treatment of the hypothyroidism suggests that this is the most likely explanation in the case presented here . success has been reported with weekly observational administration of thyroxine.3 specifically , grebe et al demonstrated through their study that once - weekly dosing was an effective means of maintaining tsh levels and thyroid hormone levels within relatively normal ranges , and did not appear to place the patient at risk of toxic complications associated with high doses of thyroxine.28 kubota et al explain that with this type of arrangement , the patient maintains their status as a patient , the hypothyroid condition is treated , and the risks of serious complications are avoided.3 a number of studies have shown that both iron and calcium supplements can potentially decrease the absorption of thyroxine through the formation of a nonabsorbable complex in the gut.9,10 given the increased incidence of hypothyroidism in the elderly population and the association of both osteoporosis and anemia with this condition , it is important to consider the interaction of these supplements with thyroxine as a potential cause for treatment failure . to eliminate this interaction and to maximize the benefit of each treatment , other studies have shown that gastrointestinal disorders and anatomical gastrointestinal abnormalities , such as untreated celiac disease and short - gut syndrome , may affect the absorption of thyroxine.11,12 celiac disease is an important consideration in this group of patients , considering the autoimmune association between these two disorders and the frequency in which they occur in patients simultaneously . in terms of patients with celiac disease , normalization of thyroid hormone levels and tsh have been observed once a gluten - free diet has been implemented.11 normal absorption of thyroxine is also known to be affected by changes in the acidic environment of the stomach . specifically , it has been shown that patients with impaired gastric acid secretion , whether secondary to helicobacter pylori infection or atrophic gastritis , required higher doses of thyroxine - replacement therapy to become euthyroid.13 similar but reversible findings were also seen in patients treated with omeprazole , a proton - pump inhibitor used to suppress acid secretion.13 other common medications to consider in this group of patients are estrogen - replacement therapy and the selective estrogen receptor modulator raloxifene . arafah showed that a group of 18 postmenopausal women treated with thyroxine - replacement therapy demonstrated increases in serum thyroxine and thyroxine - binding globulin ( tbg ) concentrations while receiving estrogen treatment.14 however , patients with a diagnosis of hypothyroidism had decreased ft4 levels ( 22 5 pmol / l to 18 4 pmol / l ) and increased serum thyrotropin levels ( 0.9 1.1 to 3.2 3.1 ) . the coadministration of raloxifene and levothyroxine has also resulted in increased dosing requirements for thyroxine.15 it is not clear whether the increased dosing of thyroid - replacement treatment is related to an absorption problem or related to a change in the tbg level . garwood et al report a case where separation of these medications by 12 hours enabled their patient to reduce her thyroxine dosage , suggesting that it may be related to absorption , although the mechanism is unclear.15 in younger female patients , pregnancy should also be considered as a potential cause for increasing tsh levels and the need for an increase in thyroxine dosing , particularly in previously stable patients . similar to patients on estrogen - replacement therapy , the increased thyroid replacement dosing is attributed to an increase in tbg , resulting in decreased levels of ft4 and ft3 and subsequent rises in tsh levels.16 the increased thyroxine dosage requirements have been estimated at approximately 45% higher than their regular dose of thyroxine.16 once each of the various factors discussed above has been thoroughly investigated and eliminated , pseudomalabsorption of thyroxine should be considered in patients failing to achieve normal tsh despite high levels of thyroxine . this diagnosis and normal intestinal absorption can be confirmed through a supervised loading doses of up to 1 mg of oral thyroxine.5 it is often recommended that this be completed when patients are fasting , as uptake of thyroxine has been found to be greater in a fasting state.2 patients in these situations should respond with a rapid lowering of their tsh levels and an increase in ft4 levels . it has been reported that most levothyroxine absorption takes place within the first few hours of administration and that serum ft4 levels peak within 2 hours of administration.26 patients such as the one described in this case report are often emphatic about their compliance with the prescribed treatment . the obvious consequence of untreated hypothyroidism is the persistence of the common symptoms of fatigue , weight gain , constipation , and poor concentration , and the morbidity associated with each of these . however , there are more serious and potentially life - threatening consequences of hypothyroidism if left untreated . specifically , myxedema coma is a rare but serious medical emergency and definitely the most severe complication of decompensated hypothyroidism . it is often the result of coexisting medical conditions or drugs , and has been found to have a mortality rate of approximately 80% in untreated patients.1 another rare and potentially severe complication of untreated hypothyroidism is the development of a pituitary hyperplasia and hyperprolactinemia , as was seen in our patient . although there have been no documented cases where specifically the intentional noncompliance with thyroxine treatment has led to the development of pituitary hyperplasia , it is well established that one of the potential causes for hyperprolactinemia and pituitary hyperplasia is hypothyroidism.26,27 in these situations , the increase in pituitary size and elevated prolactin secretion are felt to be secondary to increased synthesis of thyroid releasing hormone ( trh ) in response to chronically low thyroid hormone levels . the development of pituitary hyperplasia in itself has the potential for quite severe complications , including headaches , neurological deficits , and visual loss . prolonged hyperprolactinemia may lead to infertility , hypopituitarism , and low bone density.28 although hypothyroidism is clearly not the only cause of a pituitary hyperplasia and elevated prolactin levels , resolution of these findings with appropriate treatment of the hypothyroidism suggests that this is the most likely explanation in the case presented here . success has been reported with weekly observational administration of thyroxine.3 specifically , grebe et al demonstrated through their study that once - weekly dosing was an effective means of maintaining tsh levels and thyroid hormone levels within relatively normal ranges , and did not appear to place the patient at risk of toxic complications associated with high doses of thyroxine.28 kubota et al explain that with this type of arrangement , the patient maintains their status as a patient , the hypothyroid condition is treated , and the risks of serious complications are avoided.3 this case highlights the serious complication of pituitary hyperplasia that may occur with prolonged and untreated hypothyroidism in the context of pseudomalabsorption of levothyroxine . recognizing pituitary hyperplasia and hyperprolactinemia as a potential complication of this disorder may also prevent the potential misdiagnosis of a prolactinoma leading to unnecessary investigations and inappropriate treatment . supervised loading of levothyroxine can help to confirm this disorder and avoid extensive and unnecessary investigations . also unique to this case report patient awareness of this serious complication and the rapid , demonstrable resolution with adequate thyroid hormone replacement may provide the motivation to comply with levothyroxine treatment .
we implement genetic algorithm based predictive model building as an alternative to the traditional stepwise regression . we then employ the information complexity measure ( icomp ) as a measure of model fitness instead of the commonly used measure of r - square . furthermore , we propose some modifications to the genetic algorithm to increase the overall efficiency .	for instance , exhaustive search becomes unreasonable as the number of variables increases ; employing a multiple regression search produces over one billion possible models for data with 30 explanatory variables . in ecological studies , one of the commonly used methods for selection is stepwise regression , with forward or backward variable selection algorithms . these methods have been criticized for lacking the ability to truly pick the best model for several reasons ( boyce et al . , 1974 ; wilkinson , 1989 ) . one problem is that the choice by which the variables enter the selection algorithm is not justified theoretically . in addition , the probabilities for the selection procedure are chosen arbitrarily , which may lead to a poorly selected model . since these methods employ local search , it is unlikely that the global maximum set of variables will be found ( mantel , 1970 ; hocking , 1976 , 1983 ; moses , 1986 ) . we propose the use of genetic algorithms ( gas ) to determine the subset of variables with the highest goodness of fit for a multiple regression model . due to their global search capabilities , the ga based model building is not prone to the problems associated with local search method , hence is a wise choice for this procedure . we now explain the basics of gas briefly ; a thorough one can be found in goldberg ( 1989 ) . genetic algorithms are a set of optimization techniques inspired by biological evolution , operating under natural selection . first developed by holland ( 1975 ) , they have grown in popularity because of the ability of the algorithm to perform well on many different types of problems . in a ga , possible solutions are coded using binary strings , which are called chromosomes . each chromosome has a fitness value associated with it based on how well the string model parameters predicts the dependent variables . during each generation , which is the time step of the algorithm , a population of chromosomes compete to have their genes the selection step is used to pick the chromosomes for the next generation based on their fitness . those selected enter the mating pool , where two chromosomes mate using crossover . during this phase , parts of each parent string are swapped to form two new chromosomes that have certain aspects of their parents . mutation occurs with a small probability and is defined by a change from 0 to 1 or 1 to 0 in the binary string . mutation allows the introduction of new genes that were either lost from the population or were not there to start with . through successive generations , increasingly better chromosomes come to dominate the population , and the optimal solution ( or something very close ) is realized . a key component of a ga is the method to evaluate the fitness of a chromosome . thus , in order to use a ga for model selection in multiple regression , a way to evaluate the chromosomes is needed . more specifically the fittest chromosome is the set of parameters that maximizes the explanatory power of the model with minimum number of parameters . bozdogan ( 1988 , 2004 ) considered complexity as a measure of fitness , which can be described as follows : the complexity of a system ( of any type ) is a measure of the degree of interdependency between the whole system and a simple enumerative composition of its subsystems or parts . the concept of information complexity was first introduced by akaike ( 1973 ) as a measure of the complexity of a model : it is a relative measure of the information lost when a given model is used , and can be described as a function of the precision and accuracy of the model . the expression for aic is given as where l(k ) denotes the maximum likelihood function , ^k is the maximum likelihood estimate of parameter vector k , and m(k ) is the number of parameters in the model . the first term of aic gives the lack of fit of the model , and the second term is a penalty for the number of parameters in the model . the model with the lowest aic value is considered the best , because the model successfully determines the underlying stochastic process with the least number of parameters . although aic does take into account the problem of over - fitting , where other measures such as r - square do not , aic is not sensitive to parameter dependency , which is an important component for model selection . if a model with both low variance and low covariance can be produced , then the parameters can be better estimated , as they will not be correlated . as an alternative to aic , we consider icomp as a complexity measure which considers variance and covariance , and accounts for the problem of over - fitting the model . it is calculated by where l(k ) again denotes the maximum likelihood function , ^k is the maximum likelihood estimate of parameter vector k under the model mk , c is a real - valued complexity measure , and ^model is the estimated covariance matrix of the parameters of the model . the main difference between the two measures of complexity is that aic only considers the number of parameters as a penalty , whereas icomp considers the covariance between parameters . in predictive model building , this value for icomp is based on the inverse - fisher information matrix ( ifim ) . for multiple regression , the value of icomp can be directly calculated after regression is implemented , and is given by n is the number of parameters in the model , q is the number of observations , ^2=sse / n , tr(^2(xx)1 ) is the trace of the observation matrix multiplied by its inverse and then scaled by ^2 , and \|^2(xx)1\| is the determinant of the previous matrix . since the model with the lowest icomp value is considered the best , the ga chooses strings biased toward those with the lowest value . a commonly used method to form the mating pool is proportional selection , which depends on selecting strings for the mating pool with a probability proportional to their fitnesses . in proportional selection , the first step of the calculation of the fitness values is subtracting the icomp value of each string in that generation from the maximum value of icomp in the population . that is , for each i = 1,2, ,n , where n is the size of the population . then the average icomp difference ( the average fitness ) for the total population is calculated as finally , each string is given a fitness value that is the ratio of its icomp difference and the average fitness of the population : here we consider the implementation of ga 's for predictive model selection and discuss possible improvements . the first step to implementing a ga for any optimization problem is to encode the input variable into binary strings . in the case of multiple linear regression we wish to fit the data to where y is an n 1 response vector , x is an n q matrix of the data points , is a q 1 coefficient matrix , and is an n 1 error vector with entries from independent normal distributions [ n(0 , ) for all components ] . the encoding is done by creating a binary string which has n + 1 bits , where each bit represents a different parameter of the model and an intercept . the last n bits correspond to the n explanatory variables contained in the dataset , whereas the first bit is the intercept for the linear model . a parameter is included in the model if the value of the bit for that parameter is a 1 and is excluded if it is a 0 . for example , suppose we have a dataset where we are interested in predicting the reproductive fitness of a species of trees . the possible explanatory variables may include : density of trees in the surrounding area , average temperature of environment , average rainfall of environment , circumference of trunk , longitude of environment , latitude of environment , prevalence of disease in environment . in this case for example , the string 10010111101 would represent a model which includes the intercept , soil ph , average temperature of environment , average rainfall of environment , circumference of trunk , longitude of environment , and prevalence of disease in environment . similarly , the string 00001000110 is a model that has no intercept , and includes density of trees in the surrounding area , longitude of environment , and latitude of environment ( see table 1 ) . the probability that a string will be chosen for the mating pool is proportional to its fitness value . note that the string with the worst icomp value will never be picked for the mating pool , as its fitness will be 0 . now that we have a method of encoding information and a method to evaluate the fitness values , we have to determine the remaining parameters of the ga . the first one we consider is the method of creating the initial population and determining its size . unless previous knowledge about the problem is given , it is commonplace in gas to randomly generate binary strings ( goldberg , 1989 ) . however , in the case of model selection , a user may want to force a parameter(s ) to be included , even if it is not part of the model with the lowest complexity . in this case , the initial population can be generated in such a way that certain parameters are always in the model . in addition to determining the method to generate the population , the user must choose the size of the initial population . generally the size should not be too large , as this will slow the algorithm , and should not be so small that genetic drift takes over the course of evolution of the population . in typical gas , the size of the population stays the same ; however , this may not be an effective use of computation . we will see in the next section that starting with a larger size then reducing it may be more effective . finally , we discuss the genetic operators which allow the algorithm to find the optimal model . first , the probability of crossover , pc , is chosen . in the mating pool , a pair of strings are chosen along with a random number from [ 0 , 1 ] . if that number is less than the probability of crossover , crossover occurs . thus , if pc = 1 , then every pair will cross , and if pc = 0 , then no strings will be altered by crossover . after the choice of pc , the number of crossover points must be chosen . then the bits from the parent strings are swapped to create two new offspring strings ( see figure 1 ) . the purpose of crossover is to bring together models which have components that reduce complexity . recall the previous example about trees , where we specified two strings , which we will call parent 1 and parent 2 . applying crossover to the two parents creates two offspring ( see figure 1 ) , where offspring 1 represents a model with an intercept , soil ph , average temperature of environment , longitude of environment , latitude of environment , and prevalence of disease in environment , and offspring 2 represents a model that includes density of trees in the surrounding area , average rainfall of environment , circumference of trunk , and longitude of environment . through successive generations and application of crossover of low complexity models , the algorithm is able to find the least complex model ( or something close to it ) to explain the data . but , what happens if the actual least complex model includes a parameter that is not present in the population , that is , the position in the string that represents the parameter is fixed at 0 ? this value gives the probability that at each location in the string the bit will be flipped . flipping is defined as a change of a 0 to 1 or a 1 to a 0 . however , strings used for other applications of ga 's are usually longer than the ones used for determining least complex models . although there are ongoing studies on determining optimal crossover and mutation rates ( such as nested gas , self - adjusting parameterless gas ) , these rates can be determined by trial and error or by pilot runs before the actual data set has been used to build a model . we conclude this section with a pseudo code for a ga used to find the least complex model that sufficiently describes the data . generate initial population while ( t < max generations or the maximum number of computations have not been executed ) ( a)calculate icomp for the model each string encodes ( b)select strings for the mating pool ( c)create a new population using crossover ( d)mutate new population while the use of a typical ga for model selection already proves to be more efficient than stepwise regression , with a few modifications , the process can show a 10-fold increase in accuracy given the same amount of computation . first , we discuss the modifications , and then we explain the study done to determine the effectiveness of these modifications . the first modification is changing how the initial population was created . according to fisher 's fundamental theorem of natural selection ( fisher , 1930 ) , the increase in mean fitness is equal to the variance in fitness . for model selection using gas , the easiest way to increase variance in fitness would be to allow every model to be represented in the population . of course , this is impossible for a model with a large number of possible explanatory variables , and would amount to doing an exhaustive search . we believe that the next best procedure is to force the population to start with the highest variance in each position of the chromosome . since each position is either a 0 or 1 , this would imply that at each position there are the same amount of 0 s and 1 s across the entire population . to implement this procedure , the other half is generated by taking each of the chromosomes in the first half and changing each bit from 1 to 0 or 0 to 1 . in addition to increasing variance at each position , this procedure guarantees that within one generation , recombination alone could generate the best model . this does not imply that mutation is not necessary , as selection acts on the entire string , not individual positions . since selection will reduce variance at each position , mutation is still required to maintain some variance . the second modification is starting with a larger initial population and then reducing it in size . we have used a reduction method that adapts to the changes in the algorithm in this study . adaptively reducing the population is done by calculating the change in the best fitness between two consecutive generations and then reducing the population based on this change . more specifically , the population is reduced by the percentage increase in best fitness up to some limit . clearly , there must be a limit to the percentage of reduction , since the population should not be reduced too much , and also because the percent change can be more than 100 . here the amount of population reduction depends on the complexity of the problem , that is the type of the fitness function ( such as mse , aic , icom , mallow 's cp and so on ) used . this limit on the reduction may be determined by pilot studies . the percent change in fitness at generation t the change is calculated by the formula ftbest=\|ft1bestft2best\|/\|ft2best\| . the population size n(t ) at each generation is given by the recursive relation when using the adaptive method , elitism was also implemented . elitism is a procedure commonly used in gas in order to pass the best chromosome , or a group of the best chromosomes , to the next generation without any modifications . using elitism guarantees that the change in best fitness is always non - negative . as a result , since we wished to minimize icomp , we set the fitness of each chromosome to be the negative of the icomp value . the choices of these parameters should be done by considering characteristics of the problem such as the expected increase in fitness over time . this is typically a difficult characteristic to determine . generally , as the number of variables increase , the value of fmaxbest should decrease . as the number of variables increases , so does the number of possible values of icomp , and the likelihood that the population will evolve slower . the value of min_popsize should be chosen so that it is quite small ( 5 ) , regardless of the number of variables . as a side note , the ga with no population reduction is a special case of the adaptive method where fmaxbest=0 . the final modification to a ga for multiple regressions is the use of binary tournament instead of proportional selection . in this selection scheme , two chromosomes are chosen at random , and the one with the lower icomp value is selected for the mating pool . then both chromosomes are put back into the pool of contestants of the tournament . one advantage of this technique is that icomp values need only be calculated for the chromosomes that participate in the tournament . for models with few explanatory variables , this gain in computation may be negligible . on the other hand , for those models with many variables , the reduction in computation means that more generations can be used , or the initial population can be larger . when the population is being reduced , genetic drift may be amplified , since the sampling space for the next generation decreases . proportional selection may increase this effect because a few chromosomes with extremely high fitness are expected to be picked often for the mating pool . however , selection to participate in the tournament is random , avoiding the over - selection of chromosomes with extremely large fitness values . to test the benefits of these modifications , we used the data set in bozdogan ( 2004 ) where the predictive model is constructed for body fat and 13 explanatory variables . in order to determine how well the ga was performing , all subsets of the variables ( 2 1 = 16,383 subsets ) were used to generate a model , and then the icomp value was determined . testing was done to ensure the same icomp values were being generated for the matlab and java code . these cases differed in the value of fmaxbest , and as a result in the initial population size . all trials were allowed 600 computations , where a computation is the total number of chromosomes summed over every generation . each different ga scheme ran through 200 trials and the number of times the correct model was selected was recorded . parameters that were the same for all genetic algorithm schemes . the frequency of the correct model being selected over 200 trials . the first step to implementing a ga for any optimization problem is to encode the input variable into binary strings . in the case of multiple linear regression we wish to fit the data to where y is an n 1 response vector , x is an n q matrix of the data points , is a q 1 coefficient matrix , and is an n 1 error vector with entries from independent normal distributions [ n(0 , ) for all components ] . the encoding is done by creating a binary string which has n + 1 bits , where each bit represents a different parameter of the model and an intercept . the last n bits correspond to the n explanatory variables contained in the dataset , whereas the first bit is the intercept for the linear model . a parameter is included in the model if the value of the bit for that parameter is a 1 and is excluded if it is a 0 . for example , suppose we have a dataset where we are interested in predicting the reproductive fitness of a species of trees . the possible explanatory variables may include : density of trees in the surrounding area , average temperature of environment , average rainfall of environment , circumference of trunk , longitude of environment , latitude of environment , prevalence of disease in environment . in this case for example , the string 10010111101 would represent a model which includes the intercept , soil ph , average temperature of environment , average rainfall of environment , circumference of trunk , longitude of environment , and prevalence of disease in environment . similarly , the string 00001000110 is a model that has no intercept , and includes density of trees in the surrounding area , longitude of environment , and latitude of environment ( see table 1 ) . the probability that a string will be chosen for the mating pool is proportional to its fitness value . note that the string with the worst icomp value will never be picked for the mating pool , as its fitness will be 0 . now that we have a method of encoding information and a method to evaluate the fitness values , we have to determine the remaining parameters of the ga . the first one we consider is the method of creating the initial population and determining its size . unless previous knowledge about the problem is given , it is commonplace in gas to randomly generate binary strings ( goldberg , 1989 ) . however , in the case of model selection , a user may want to force a parameter(s ) to be included , even if it is not part of the model with the lowest complexity . in this case , the initial population can be generated in such a way that certain parameters are always in the model . in addition to determining the method to generate the population , the user must choose the size of the initial population . generally the size should not be too large , as this will slow the algorithm , and should not be so small that genetic drift takes over the course of evolution of the population . in typical gas , the size of the population stays the same ; however , this may not be an effective use of computation . we will see in the next section that starting with a larger size then reducing it may be more effective . finally , we discuss the genetic operators which allow the algorithm to find the optimal model . first , the probability of crossover , pc , is chosen . in the mating pool , a pair of strings are chosen along with a random number from [ 0 , 1 ] . thus , if pc = 1 , then every pair will cross , and if pc = 0 , then no strings will be altered by crossover . after the choice of pc , the number of crossover points must be chosen . then the bits from the parent strings are swapped to create two new offspring strings ( see figure 1 ) . the purpose of crossover is to bring together models which have components that reduce complexity . recall the previous example about trees , where we specified two strings , which we will call parent 1 and parent 2 . applying crossover to the two parents creates two offspring ( see figure 1 ) , where offspring 1 represents a model with an intercept , soil ph , average temperature of environment , longitude of environment , latitude of environment , and prevalence of disease in environment , and offspring 2 represents a model that includes density of trees in the surrounding area , average rainfall of environment , circumference of trunk , and longitude of environment . through successive generations and application of crossover of low complexity models , the algorithm is able to find the least complex model ( or something close to it ) to explain the data . but , what happens if the actual least complex model includes a parameter that is not present in the population , that is , the position in the string that represents the parameter is fixed at 0 ? this value gives the probability that at each location in the string the bit will be flipped . flipping is defined as a change of a 0 to 1 or a 1 to a 0 . however , strings used for other applications of ga 's are usually longer than the ones used for determining least complex models . although there are ongoing studies on determining optimal crossover and mutation rates ( such as nested gas , self - adjusting parameterless gas ) , these rates can be determined by trial and error or by pilot runs before the actual data set has been used to build a model . we conclude this section with a pseudo code for a ga used to find the least complex model that sufficiently describes the data . generate initial population while ( t < max generations or the maximum number of computations have not been executed ) ( a)calculate icomp for the model each string encodes ( b)select strings for the mating pool ( c)create a new population using crossover ( d)mutate new population while the use of a typical ga for model selection already proves to be more efficient than stepwise regression , with a few modifications , the process can show a 10-fold increase in accuracy given the same amount of computation . first , we discuss the modifications , and then we explain the study done to determine the effectiveness of these modifications . the first modification is changing how the initial population was created . according to fisher 's fundamental theorem of natural selection ( fisher , 1930 ) , the increase in mean fitness is equal to the variance in fitness . for model selection using gas , the easiest way to increase variance in fitness would be to allow every model to be represented in the population . of course , this is impossible for a model with a large number of possible explanatory variables , and would amount to doing an exhaustive search . we believe that the next best procedure is to force the population to start with the highest variance in each position of the chromosome . since each position is either a 0 or 1 , this would imply that at each position there are the same amount of 0 s and 1 s across the entire population . to implement this procedure , the other half is generated by taking each of the chromosomes in the first half and changing each bit from 1 to 0 or 0 to 1 . in addition to increasing variance at each position , this procedure guarantees that within one generation , recombination alone could generate the best model . this does not imply that mutation is not necessary , as selection acts on the entire string , not individual positions . since selection will reduce variance at each position , mutation is still required to maintain some variance . the second modification is starting with a larger initial population and then reducing it in size . we have used a reduction method that adapts to the changes in the algorithm in this study . adaptively reducing the population is done by calculating the change in the best fitness between two consecutive generations and then reducing the population based on this change . more specifically , the population is reduced by the percentage increase in best fitness up to some limit . clearly , there must be a limit to the percentage of reduction , since the population should not be reduced too much , and also because the percent change can be more than 100 . here the amount of population reduction depends on the complexity of the problem , that is the type of the fitness function ( such as mse , aic , icom , mallow 's cp and so on ) used . this limit on the reduction may be determined by pilot studies . the percent change in fitness at generation t is denoted by ftbest and the limit is denoted by fmaxbest . the population size n(t ) at each generation is given by the recursive relation when using the adaptive method , elitism was also implemented . elitism is a procedure commonly used in gas in order to pass the best chromosome , or a group of the best chromosomes , to the next generation without any modifications . using elitism guarantees that the change in best fitness is always non - negative . as a result , the population never increases in size . since we wished to minimize icomp , we set the fitness of each chromosome to be the negative of the icomp value the choices of these parameters should be done by considering characteristics of the problem such as the expected increase in fitness over time . this is typically a difficult characteristic to determine . generally , as the number of variables increase , the value of fmaxbest should decrease . as the number of variables increases , so does the number of possible values of icomp , and the likelihood that the population will evolve slower . the value of min_popsize should be chosen so that it is quite small ( 5 ) , regardless of the number of variables . as a side note , the ga with no population reduction is a special case of the adaptive method where fmaxbest=0 . the final modification to a ga for multiple regressions is the use of binary tournament instead of proportional selection . in this selection scheme , two chromosomes are chosen at random , and the one with the lower icomp value is selected for the mating pool . then both chromosomes are put back into the pool of contestants of the tournament . one advantage of this technique is that icomp values need only be calculated for the chromosomes that participate in the tournament . for models with few explanatory variables , this gain in computation may be negligible . on the other hand , for those models with many variables , the reduction in computation means that more generations can be used , or the initial population can be larger . when the population is being reduced , genetic drift may be amplified , since the sampling space for the next generation decreases . proportional selection may increase this effect because a few chromosomes with extremely high fitness are expected to be picked often for the mating pool . however , selection to participate in the tournament is random , avoiding the over - selection of chromosomes with extremely large fitness values . to test the benefits of these modifications , we used the data set in bozdogan ( 2004 ) where the predictive model is constructed for body fat and 13 explanatory variables . in order to determine how well the ga was performing , all subsets of the variables ( 2 1 = 16,383 subsets ) were used to generate a model , and then the icomp value was determined . testing was done to ensure the same icomp values were being generated for the matlab and java code . these cases differed in the value of fmaxbest , and as a result in the initial population size . all trials were allowed 600 computations , where a computation is the total number of chromosomes summed over every generation . each different ga scheme ran through 200 trials and the number of times the correct model was selected was recorded . parameters that were the same for all genetic algorithm schemes . the frequency of the correct model being selected over 200 trials . while model selection remains to be a difficult procedure in case of a large number of parameters , using a ga to find the least complex model can be quite helpful . we have shown that our modifications to the original ga for model selection can yield strong results . additionally , the ga approach ( because of the use of icomp ) is better at handling data in which collinearity exist than the traditional selection methods such as forward , backward , and stepwise selection . in particular it is clear that the modifications had a large effect on the accuracy of the ga . this seems to indicate that we may reduce computation and still get statistically the same accuracy if we employ diversification . in all trials , along with the facts presented above and the fact that diversification is easy ( and not costly ) to implement , it is our recommendation that it be used for model selection using gas . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .
congenital hypotrichosis may be due to a number of causes and may have multiple systemic associations . a child born of second - degree consanguineous marriage was found to have monilethrix as the cause of congenital hypotrichosis . a detailed systemic evaluation in the child revealed atrial septal defect and a hypoplastic right thumb leading to a diagnosis of coexisting holt - oram syndrome .	monilethrix is an autosomal dominant disorder of hair shaft with regular thinning of hair shaft with fracture of the hair shaft at constricted points , which is commonly caused due to human hair keratin hhb6 gene located on long arm of chromosome 12 . patients have short and fragile hair not requiring haircut since childhood commonly over the occipital and temporal scalp , but may also involve eyebrows , eyelashes , axillary , pubic or body hair . holt - oram syndrome is a syndrome characterized by limb defects associated with cardiovascular anomalies . it is an autosomal dominant disorder caused due to mutations of tbx5 gene located on a long arm of chromosome 12 . the cardiovascular defects are usually in the form of septal defects commonly atrial septal defect . limb defects range from a hypoplastic thumb , metacarpals , carpals , radius to an absent limb . a 2-month - old female child , first born of second degree consanguineous marriage was brought with complaints of absence of hair over the scalp , eyebrows and body since birth . examination revealed absence of hair from the scalp , eyebrows and body with presence of hair over eyelashes [ figure 1 ] . other examination findings included a hypoplastic thumb on the right side with normal nails [ figure 2 ] . clinical photograph showing absence of hair over scalp and eyebrows clinical photograph showing hypoplastic thumb dermoscopic examination revealed presence of numerous empty follicles , small broken hairs , black dots and hair with uniform nodal dilatations with intermittent constrictions at which there was shaft breakage . dermoscopy of scalp showing hairs with monilethrix microscopic hair shaft examination could not be carried out as hair were too short for plucking . echocardiography showing atrial septal defect thus , a diagnosis of holt - oram syndrome with monilethrix was arrived at . it can be transmitted as an isolated condition , which has an autosomal dominant inheritance . it can also be associated with papular atrichia , keratosis pilaris , vitamin d resistant rickets , ectodermal dysplasia , moynahan 's syndrome , hypotrichosis simplex , marie - unna hereditary hypotrichosis , pseudo - thalidomide syndrome , amino acid metabolism and hair shaft disorders . the common hair shaft disorders , which may present as hypotrichosis include monilethrix , pili torti and pili annulati . monilethrix ( omim # 158000 ) is an uncommon hair shaft defect , which can manifest as congenital hypotrichosis . monilethtix is associated with trichorrhexis nodosa , nail and teeth defects , retarded growth and juvenile cataracts . the conditions where a hypoplastic thumb can be associated with atrial septal defect include holt - oram syndrome and vacterl association . holt - oram syndrome ( omim # 142900 ) is associated with limb defects such as hypoplastic thumb , metacarpals , carpals or radius defects and cardiovascular anomalies especially septal defects . vacterl association includes vertebral anomalies , anal atresia , cardiovascular anomalies , especially septal defects , trachea - esophageal fistula , renal defects and limb defects like hypoplastic thumb , syndactyly , polydactyly and forearm bony defects like radial aplasia . the presentation of monilethrix as congenital hypotrichosis is not very common . in this patient , the association of holt - oram syndrome with monilethrix , in spite of the causative genetic mutations being on the same chromosome , has not been reported to the best of our knowledge .
backgroundfew studies have investigated differences in age- and gender - specific educational gradients in tobacco smoking among the whole range of adult age groups . we examined educational inequality in smoking among japanese adults aged 2594 years.methodsusing a large nationally representative sample ( 167,925 men and 186,588 women ) in 2010 , prevalence of current smoking and heavy smoking among daily smokers and their inequalities attributable to educational attainment were analyzed according to sex and age groups.resultsamong men aged 2534 years , junior high school graduates had the highest current smoking prevalence at 68.4% ( 95% confidence interval [ ci ] , 66.0%70.6% ) , and graduate school graduates had the lowest at 19.4% ( 95% ci , 17.2%21.9% ) . high school graduates had the second highest current smoking prevalence ( e.g. , 55.9% ; 95% ci , 54.9%56.8% in men aged 2534 years ) . among men aged 7594 years , the difference in current smoking across educational categories was small . a similar but steeper educational gradient in current smoking was observed among women . among women aged 2534 years , junior high school graduates had the highest current smoking prevalence at 49.3% ( 95% ci , 46.3%52.3% ) , and graduate school graduates had the lowest at 4.8% ( 95% ci , 2.9%7.4% ) . compared with older age groups , such as 6594 years , younger age groups , such as 2554 years , had higher estimates of inequality indicators for educational inequality in both current and heavy smoking in both sexes.conclusionseducational inequalities in current and heavy smoking were apparent and large in the young population compared with older generations . the current study provides basic data on educational inequalities in smoking among japanese adults .	tobacco smoking is the most attributable and preventable risk factor for adult mortality and morbidity in japan.1 , 2 tobacco smoking has been confirmed as an independent risk factor for many disorders , such as cancer and cardiovascular diseases , with dose - response verifications.3 , 4 heavy smokers are more likely to suffer from tobacco - related harm than light smokers . smoking cessation and tackling regional and socioeconomic inequalities in smoking are key public health targets throughout the world . the world health organization ( who ) 's commission on social determinants of health recommended , in its final report , monitoring and evaluating socioeconomic inequalities in health and health behavior , including smoking . japan 's new health promotion strategy , health japan 21 ( second term ) , follows the who recommendations and includes monitoring socioeconomic inequalities in tobacco smoking and other public health targets . when monitoring socioeconomic inequality in smoking , using educational attainment as a socioeconomic indicator is important . educational attainment is a representative socioeconomic factor that barely changes in adulthood after around 25 years of age , whereas other socioeconomic variables , such as income and occupation , could change considerably during a life - course . moreover , educational attainment could reflect key determinants of the initiation and habituation of smoking and other health behaviors , such as health literacy . although some japanese studies have investigated smoking inequalities according to socio - economic factors , such as income and occupation,9 , 10 no study has examined smoking according to education in japan . recent european studies have consistently shown higher prevalence of tobacco smoking among poorly educated populations , whereas patterns of smoking prevalence in terms of sex and age groups vary across regions . although there is relatively rich evidence from western countries , data from asian regions are scarce . moreover , data on educational inequality in smoking in older persons , especially those aged 75 years or more , are scarce even worldwide.3 , 11 to start continuous monitoring of educational inequality in health , in 2010 , the comprehensive survey of living conditions of people on health and welfare ( cslc ) , a large nationally representative population - based cross - sectional survey in japan , collected information on education , in addition to health behavior indicators , including smoking status . thus , our objective in this study was to investigate the magnitude of educational inequality in smoking and the prevalence of current and heavy smoking according to sex , age , and education among japanese adults . utilizing the large nationally representative dataset , we sought to provide detailed evaluations of age- and sex - specific variations in smoking inequality in japan , covering the whole range of adult age groups from 25 to 94 years old . we used data from a nationally representative cross - sectional survey : the 2010 cslc , conducted by the japanese ministry of health , labour and welfare ( mhlw ) . out of 940,000 inhabited census tracts ( the sampling unit for national census in 2005 ) , 5510 were randomly sampled across japan in 2010 for the collection of data from all household members within each census tract . data were used with permission from mhlw . the study was reviewed and approved by the research ethics committee of the osaka medical center for cancer and cardiovascular diseases . levels of completed education were categorized as six groups : junior high school ( 9 years of mandatory education ) was defined as persons who graduated junior high school without graduating high school ; high school ( 12 years of education ) was defined as persons who graduated high school without graduating further educational steps ; technical school ( 1019 years of education ) 2-year college ( 14 years of education ) was defined as persons who graduated 2-year college without going to 4-year college ; 4-year university ( 16 years of education ) was defined as persons who graduated 4-year university without going to graduate school ; graduate school ( 1722 years of education ) was defined as persons who graduated graduate school having previously graduated 4-year university . current smokers were those who smoked cigarettes regularly at the time of survey , including daily and sometimes smokers . among daily smokers ( 93.8% of the current smokers in the data ) , heavy smokers were those who smoked more than 20 cigarettes per day . we analyzed japanese adults aged 2594 years because education status was less likely to change after 25 years of age . we compared current smoking prevalence and heavy smoking proportions among daily smokers according to sex , age , and education group . the percentages are shown with 95% confidence intervals ( cis ) calculated by the wald method . to show the summarized relationship between education and smoking , the age - adjusted smoking prevalence for young and middle - aged adults ( 2564 years ) was also calculated via the direct standardization method using population figures from the 2010 japanese census . following recent recommendations , to evaluate educational inequality in current and heavy smokers , we calculated multiple health disparity indicators , including absolute indicators ( rate difference and between - group variance ) and relative indicators ( rate ratio , index of disparity , and mean log deviation),15 , 16 , 17 using hdcalc software , version 1.2.4 ( the national cancer institute , rockville , md , usa ) . detailed explanations of these indicators are given in the supplementary data ( eappendix 1 ) and elsewhere.19 , 20 population weight was used to calculate measures of inequality , because the population size differed according to the education categories , reflecting educational distributions . the proportion of highly educated people in the general population has been increasing over time . such a demographic shift has an impact on population health and needs to be considered for the assessment of inequalities . inequality measures of index of disparity , mean log deviation , and between - group variance accounted for the population size of the groups in the calculation . subject numbers according to sex , age , and education group are shown in etable 1 ( for current smoking prevalence ) and etable 2 ( for heavy smoking prevalence among daily smokers ) . to maintain precision of estimates , we did not calculate the smoking prevalence ( proportion ) of the groups that included fewer than 100 subjects . although there are no convincing criteria for the sample size cut - off point , we chose a sample size of 100 based on statistical considerations of the relationship between sample size and precision . according to machin et al , the width of a 95% ci depends on the size of the point estimate . however , a sample size of 100 can maintain at least 20% point width of the 95% ci regardless of the size of the point estimates . this choice of sample size cut - off resulted in some groups for which we did not estimate smoking prevalence ( proportion ) . we evaluated the educational inequality in smoking where smoking prevalence ( proportion ) data were available for at least three education groups . all statistical analyses , except for the inequality index calculation , were performed using sas version 9.3 ( sas institute inc . , cary , nc , usa ) . we used data from a nationally representative cross - sectional survey : the 2010 cslc , conducted by the japanese ministry of health , labour and welfare ( mhlw ) . out of 940,000 inhabited census tracts ( the sampling unit for national census in 2005 ) , 5510 were randomly sampled across japan in 2010 for the collection of data from all household members within each census tract . data were used with permission from mhlw . the study was reviewed and approved by the research ethics committee of the osaka medical center for cancer and cardiovascular diseases . levels of completed education were categorized as six groups : junior high school ( 9 years of mandatory education ) was defined as persons who graduated junior high school without graduating high school ; high school ( 12 years of education ) was defined as persons who graduated high school without graduating further educational steps ; technical school ( 1019 years of education ) was defined as persons who graduated technical professional school without going to college ; 2-year college ( 14 years of education ) was defined as persons who graduated 2-year college without going to 4-year college ; 4-year university ( 16 years of education ) was defined as persons who graduated 4-year university without going to graduate school ; graduate school ( 1722 years of education ) current smokers were those who smoked cigarettes regularly at the time of survey , including daily and sometimes smokers . among daily smokers ( 93.8% of the current smokers in the data ) , we analyzed japanese adults aged 2594 years because education status was less likely to change after 25 years of age . we compared current smoking prevalence and heavy smoking proportions among daily smokers according to sex , age , and education group . the percentages are shown with 95% confidence intervals ( cis ) calculated by the wald method . to show the summarized relationship between education and smoking , the age - adjusted smoking prevalence for young and middle - aged adults ( 2564 years ) was also calculated via the direct standardization method using population figures from the 2010 japanese census . following recent recommendations , to evaluate educational inequality in current and heavy smokers , we calculated multiple health disparity indicators , including absolute indicators ( rate difference and between - group variance ) and relative indicators ( rate ratio , index of disparity , and mean log deviation),15 , 16 , 17 using hdcalc software , version 1.2.4 ( the national cancer institute , rockville , md , usa ) . detailed explanations of these indicators are given in the supplementary data ( eappendix 1 ) and elsewhere.19 , 20 population weight was used to calculate measures of inequality , because the population size differed according to the education categories , reflecting educational distributions . the proportion of highly educated people in the general population has been increasing over time . such a demographic shift has an impact on population health and needs to be considered for the assessment of inequalities . inequality measures of index of disparity , mean log deviation , and between - group variance accounted for the population size of the groups in the calculation . subject numbers according to sex , age , and education group are shown in etable 1 ( for current smoking prevalence ) and etable 2 ( for heavy smoking prevalence among daily smokers ) . to maintain precision of estimates , we did not calculate the smoking prevalence ( proportion ) of the groups that included fewer than 100 subjects . although there are no convincing criteria for the sample size cut - off point , we chose a sample size of 100 based on statistical considerations of the relationship between sample size and precision . according to machin et al , the width of a 95% ci depends on the size of the point estimate . however , a sample size of 100 can maintain at least 20% point width of the 95% ci regardless of the size of the point estimates . this choice of sample size cut - off resulted in some groups for which we did not estimate smoking prevalence ( proportion ) . we evaluated the educational inequality in smoking where smoking prevalence ( proportion ) data were available for at least three education groups . all statistical analyses , except for the inequality index calculation , were performed using sas version 9.3 ( sas institute inc . , cary , nc , usa ) . table 1 shows the current smoking prevalence according to sex , age , and education group in japan . among men aged 2534 years , junior high school graduates had highest current smoking prevalence at 68.4% ( 95% ci , 66.0%70.6% ) , and graduate school graduates had the lowest at 19.4% ( 95% ci , 17.2%21.9% ) . these figures were lower in the higher age groups : among men aged 6574 years , the corresponding figures were 27.6% ( 95% ci , 26.7%28.6% ) and 12.2% ( 95% ci , 7.9%17.7% ) for junior high school graduates and graduate school graduates , respectively . among men aged 7594 years , the differences in current smoking across educational categories were small , though we did not calculate the smoking prevalence of graduate school graduates in that age group because of the small sample size . a similar but steeper educational gradient in current smoking was observed among women . among women aged 2534 years , junior high school graduates had the highest current smoking prevalence at 49.3% ( 95% ci , 46.3%52.3% ) , and graduate school graduates had the lowest at 4.8% ( 95% ci , 2.9%7.4% ) . age - adjusted rates of young and middle - aged adults ( 2564 years ) also showed a similar gradient among both sexes . the inequality indicators for educational inequality in current smoking according to sex and age groups are shown in fig . 1 ( corresponding values are shown in etable 3 ) . compared with older age groups , such as 6594 years , younger age groups , such as 2554 years , had higher estimates of inequality indicators for educational inequality in smoking among both sexes , except for one outlier of the index of disparity for women aged 7584 years . table 2 shows the prevalence of heavy smoking ( % ) among daily smokers according to sex , age , and education groups . among both men and women aged 2544 years , prevalence of heavy smoking was highest among junior high school graduates ; for example , among men aged 2534 years , 27.7% ( 95% ci , 25.0%30.5% ) of daily smokers who were junior high school graduates were heavy smokers , while the prevalence was 13.6% ( 95% ci , 9.0%19.4% ) among graduate school graduates . heavy smokers were most prevalent among daily smokers aged 5564 years in both men ( 33.4% ) and women ( 12.1% ) . 2 shows the inequality indicators for educational inequalities in heavy smoking among daily smokers ( corresponding values are shown in etable 4 ) . compared with younger age groups , older age groups , especially those aged 5584 years for men and 4564 years for women , had lower educational inequality in heavy smoking . we found that educational inequalities in current and heavy smoking were apparent and large in the young population compared with older generations ; these disparities were consistently shown using multiple indicators of inequality . the inequality in smoking among young women is strikingly large , calling for urgent political measures to address this disparity . among those aged 2544 years , junior high school graduates had considerably higher current and heavy smoking prevalence than other education groups : e.g. , nearly half of young women who were junior high school graduates currently smoked ( 49.3%47.5% ) , a rate which is dramatically higher than other education groups . heavy smoking prevalences were the highest among those aged 5564 years in both men and women , but the educational inequalities in heavy smoking in that group were smaller than in other age groups . first , education may capture the social class of younger generations more sharply than older generations . as in most other developed countries , junior high school graduates are a minority in the younger generations in japan . they have a definite disadvantage in earning power , the job market , and having partners . the less educated among the young generations are likely to lose self - efficacy , self - stigmatize , suffer chronic psychosocial stress , and have a strong sense of relative deprivation , which may cause smoking.22 , 23 among older generations , on the other hand , graduating only elementary school or junior high school was common . for example , being a junior high school graduate might not mean being disadvantaged for older age groups , especially those over 75 years old . second , the large educational gradient in smoking among the young may reflect inequality in the initiation of smoking . among men , when currently old people were young , smoking was a more common practice , and its health risks were less acknowledged . until the 1970s , nearly 80% of japanese men smoked.24 , 25 although data on educational inequality in smoking in those periods are not available , it is plausible that highly educated people continued to smoke more in those periods than current years . third , the opportunities to quit smoking are fewer among younger and less educated people . although the present study indicated a snapshot of smoking in 2010 , an individual 's smoking trajectory is related to the accumulation of social disadvantage over the entire life - course.3 , 26 less educated smokers may be more likely to fail at quitting and to become more addicted ; thus , educational inequality in smoking among the young generation may be large . further , those who had few opportunities to quit smoking , such as partner 's and/or social cessation supports , and smoking - related diseases onset ( as an opportunity to quit ) through their life course , might smoke currently when they became old . moreover , since mortality is higher among the lower socioeconomic group , older people in the low - education group may be in the selected population ( survivors ) who tend to be non - smokers / ex - smokers . multiple factors other than education throughout a life - course may be associated with smoking , especially in a geriatric population ; therefore , educational inequality among the old population is small . the strength of this study is the provision of data with fine resolution in terms of age groups and educational attainments ; previous studies on educational inequality in smoking prevalence have used only four or fewer categories for both educational attainments and age groups.3 , 11 in the present study , the large sample size enabled us to analyze sex- and age - specific data on educational inequality in smoking using seven age groups encompassing participants 2594 years old and six educational levels from junior high school graduates to graduate school graduates . data on the geriatric population is especially valuable because such data are scarce worldwide.3 , 11 our findings are consistent with studies from the western region of the world showing an inverse association between educational attainments and smoking prevalence . however , the age- and sex - specific trends have variations across countries or with existing data . the american surgeon general report in 2014 reported educational gradients in smoking using four education levels among adults aged 18 years or older : 31.5% of adults ( 36.2% of men and 26.5% of women ) who had education of less than high school smoked currently , compared with 10.4% of college graduates ( 11.1% for men and 9.7% for women ) . in europe , an inverse association of education with smoking was also seen , especially among men in northern countries . for men , studies in ireland and the united kingdom showed inequalities in all generations ; studies in finland , denmark , germany , belgium , and spain showed inequalities among middle - aged and younger men ; and studies in other countries only showed inequalities among young adults . lag time in educational inequalities in smoking between northern and southern countries , and also a lag between women and men , were observed in the late 1990s to early 2000s in europe and interpreted as smoking epidemic patterns ( within country ) : first , male smoking prevalence increases , and then female smoking prevalence increases . however , female smoking in most asian and african countries is uncommon . in the present study the study may contribute to a better understanding of socioeconomic patterns in the smoking epidemic in an asian region , because the female smoking pattern in asia is considered to be different from europe . first , the smoking variables were self - reported without biomarker validation ; however , the quality of self - reported smoking has been noted to be high.27 , 28 nevertheless , we could not exclude the possibility that reporting error occurred and that it may be differential according to education . second , this is a cross - sectional study , so we could not refer to causality between education and smoking , although both directions of causality were assumed . we compared calculated values of inequality indices between different age groups to evaluate the magnitude of educational inequality in smoking . because evaluable levels of education were different among heavy smokers according to age group , the evaluation of the inequality would result in an underestimation among old population with fewer evaluable education levels ( due to sample size ) . furthermore , graduate school graduates showed the lowest prevalence in most sex and age categories , and their sample size was relatively small , especially among the old and among women . because some inequality indicators were calculated using the lowest smoking prevalence with a wide ci , these indicators may be unstable . fourth , we need to explain the nature of inequality indicators ; inequality indicators were calculated using the smoking prevalence sequence of highest to lowest across education categories . however , the educational rank sequence was not considered in the calculation of inequality indicators . the current study provided basic data on the educational inequalities in smoking among japanese adults , which contributes to the existing literature examining smoking inequalities according to other socio - economic positions , such as income and occupation.9 , 10 educational inequalities in smoking , especially among young adults , have been observed in japan as well as the rest of the world.3 , 11 in japan , health japan 21 ( second term ) asks for a reduction of health inequality , including smoking inequality . to achieve this goal , given the findings of this study several tobacco control measures , such as tobacco taxation and media campaigns , may possibly reduce smoking inequality due to socioeconomic factors and age,26 , 29 , 30 , 31 , 32 because the tobacco price is considered to be very low in japan according to the affordability index and anti - tobacco television media campaigns have not been conducted by the japanese government . we need to continue to monitor smoking prevalence and inequality and implement effective tobacco control measures to reduce smoking inequalities . first , the smoking variables were self - reported without biomarker validation ; however , the quality of self - reported smoking has been noted to be high.27 , 28 nevertheless , we could not exclude the possibility that reporting error occurred and that it may be differential according to education . second , this is a cross - sectional study , so we could not refer to causality between education and smoking , although both directions of causality were assumed . we compared calculated values of inequality indices between different age groups to evaluate the magnitude of educational inequality in smoking . because evaluable levels of education were different among heavy smokers according to age group , the evaluation of the inequality would result in an underestimation among old population with fewer evaluable education levels ( due to sample size ) . furthermore , graduate school graduates showed the lowest prevalence in most sex and age categories , and their sample size was relatively small , especially among the old and among women . because some inequality indicators were calculated using the lowest smoking prevalence with a wide ci fourth , we need to explain the nature of inequality indicators ; inequality indicators were calculated using the smoking prevalence sequence of highest to lowest across education categories . however , the educational rank sequence was not considered in the calculation of inequality indicators . the current study provided basic data on the educational inequalities in smoking among japanese adults , which contributes to the existing literature examining smoking inequalities according to other socio - economic positions , such as income and occupation.9 , 10 educational inequalities in smoking , especially among young adults , have been observed in japan as well as the rest of the world.3 , 11 in japan , health japan 21 ( second term ) asks for a reduction of health inequality , including smoking inequality . to achieve this goal , given the findings of this study , tobacco control measures should focus more on younger generations with low educational attainment . several tobacco control measures , such as tobacco taxation and media campaigns , may possibly reduce smoking inequality due to socioeconomic factors and age,26 , 29 , 30 , 31 , 32 because the tobacco price is considered to be very low in japan according to the affordability index and anti - tobacco television media campaigns have not been conducted by the japanese government . we need to continue to monitor smoking prevalence and inequality and implement effective tobacco control measures to reduce smoking inequalities .
objectives : the aim of this study was to histologically compare the regenerative properties of two allografts manufactured by two iranian companies.materials and methods : in this study , four 8-mm defects were produced in the calvaria of 12 rabbits . in three defects , three types of allografts namely itb , cenobone and grafton were placed and one defect served as control . samples were prepared and histomorphometric evaluations were carried out after healing periods of four weeks ( interval 1 ) and eight weeks ( interval 2 ) . qualitative and quantities variables were compared and analyzed with spss software.results:mild inflammation was observed in 45% and 12.5% of the samples in the first and second intervals , respectively . foreign body reaction was observed in only 5% of the samples . the quality of regenerated bone was immature , mixed and lamellar in 54.5% , 15.9% and 4.5% of the samples , respectively . the rate of allograft resorption was the highest and lowest in the cenobone and grafton samples , respectively . the mean amount of regenerated bone was higher in areas containing grafton ; however , the differences were not statistically significant.conclusion:despite the differences in the numerical values of bone regeneration , there were no statistically significant differences in bone generation among the material groups , and allografts manufactured in iran can be suitable alternatives to grafton with the same good properties . further studies are necessary to clarify the efficacy of these allografts .	autogenous bone is considered the best choice and the gold standard due to the presence of viable cells and its osteogenic potential . use of the host 's osseous tissue requires an additional surgical procedure and has some limitations ; in addition , it can not supply adequate graft material in multiple and extensive defects . on the other hand , unwanted consequences ( 810% ) and root resorption may occur during or after harvesting of autogenous bone , and many patients do not allow manipulation of other parts of their body to harvest grafts . allografts are human - derived materials , which have the properties of autografts but are acellular . they resemble human tissues more than other animal - derived graft materials or synthetic materials . allografts are abundantly available in tissue banks and are inexpensive ; they are sterilized and stored and can be used in pure form or in combination with autogenous bone or other materials . allografts contain growth factors and osteogenic proteins such as bone morphogenetic proteins ( bmps ) with possible osteoinductive properties [ 7 , 8 ] . the major problem associated with the use of allografts is the potential of virus transmission . the use of allografts in orthopedics was first reported in 1908 and then many studies reported the use of many types of allografts . the aim of the present study was to evaluate and compare the regenerative potential of these allografts . after obtaining ethical approval from the animal studies ethics committee of the implant research center , tehran university of medical sciences , 12 mature male white new zealand rabbits with a mean weight of 2.5 g were used in this study by observing all the ethical principles . the animals were kept for two weeks in the animal center for acclimatization under standard and similar dietary and environmental conditions . the surgical area was disinfected using 10% povidone - iodine solution and the hair in the animals head was shaved . a 78-cm straight incision was made in the midline in an antero - posterior direction ; then two lateral mucocutaneous flaps were elevated to expose the area and gain access to the parietal bone . a trephine bur , which measured 8 mm in internal diameter in a rotary handpiece under irrigation and cooling with saline solution was used to create four round symmetrical bicortical defects with 8 mm diameter and almost 1 mm depth at the two sides of the calvarium midline . because of low thickness of the cortical plate and the possibility of rupture of the brain membrane , care was exercised to prevent traumatizing the meningeal membrane . one of the defects was filled with itb allograft manufactured by the iranian tissue bank ( iranian tissue bank research and preparation center , tehran , iran ) , the second defect was filled with cenobone allograft manufactured by hamanand saz baft kish company ( trc corporation , kish , iran ) ; the third defect was filled with grafton manufactured by the american tissue bank ( osteotech inc . , one defect was left empty without any graft material as a control ( figs . 1 and 2 ) . four defects measuring 8 mm in diameter were created in the calvarium of each rabbit . one of the defects was not manipulated and designated as the control and the remaining three defects were filled with three different allografts . the properties of the allografts ( demineralized freeze dried bone allograft , dfdba ) used were as follows : itb / dfdba/ demineralized bone matrix ( dbm ) gel / cortico - cancellous/75125 mccenobone / dfdba /particle /cortico - cancellous /150500 mgrafton /dfdba /dbm putty / < 300 m itb / dfdba/ demineralized bone matrix ( dbm ) gel / cortico - cancellous/75125 m ccenobone / dfdba /particle /cortico - cancellous /150500 m grafton /dfdba /dbm putty / < 300 m the type of the material placed in each defect was recorded . to avoid bias in relation to the location of defects , randomization was used to place allografts in the first animal and then clockwise rotation was used to change the sites in which the allografts were placed in the other rabbits . finally , the periosteal flap was returned to its original location and the periosteum and the skin were sutured separately with resorbable 04 vicryl sutures ( supa , tehran , iran ) in two layers internally and externally . after the surgical operation , a pharmaceutical regimen consisting of subcutaneous injections of antibiotic ( 0.6 ml enrofloxacin ) and analgesic ( 0.1 ml ketoprofen daily for three days ) was administered . in addition , the animals underwent routine daily examinations to evaluate their postoperative status . during the healing period after the surgical operation , one of the animals in group one was lost ; therefore , the sample size in this group decreased to five samples . in order to sacrifice the animals the defect sites were sectioned by a saw and a notch was produced in the occipital area to assist in identifying the direction of the defects . the samples were placed in 10% formalin solution ( neutral buffered formalin ) in closed containers and submitted to the laboratory in a blind manner . the blocks were placed in 10% formalin ( sigma aldrich chemie gmbh , taufkirchen , germany ) for five days , followed by placement in 5% formic acid ( bayer ag , leverkusen , germany ) for decalcification . then , the samples were dehydrated in ethanol and immersed in paraffin . during the decalcification process , which usually takes 10 days to complete , fresh acid solution was used every day and the extent of decalcification was evaluated until it reached the desired level . at least ten 5-mm thickness cross - sections were made from each defect and all the cross - sections were carefully evaluated by two oral pathologists twice . all the cross - sections were photographed under a binocular light microscope ( bx51 , olympus , tokyo , japan ) connected to a camera ( dp25 , olympus , tokyo , japan ) . all the parameters were measured using computer software ( dp2-bsw , olympus , tokyo , japan ) . in case of disagreement , the cross - section was reevaluated by the two pathologists and the result was recorded after a consensus was reached . spss version 20 was used for statistical analysis and processing of data ( spss inc . friedman 's test was used to compare qualitative variables and also bone regeneration due to deviation from normal distribution . in cases in which friedman 's test was significant , adjusted p values statistical significance was set at p<0.05 and p values between 0.06 and 0.10 were considered as marginally significant . spss version 20 was used for statistical analysis and processing of data ( spss inc . friedman 's test was used to compare qualitative variables and also bone regeneration due to deviation from normal distribution . in cases in which friedman 's test was significant , adjusted p values statistical significance was set at p<0.05 and p values between 0.06 and 0.10 were considered as marginally significant . the inflammation severity was recorded in a descending order in the control , grafton , itb and cenobone samples , respectively . also , there were statistically significant differences in the inflammation severity between the first and second months ( p=0.02 and p=0.03 , respectively ) . maximum differences were observed between cenobone and control group in the first month and cenobone and grafton in the second month ( tables 1 and 2 ) . analysis of the data and parameters in the first month i= itb allograft manufactured by the iranian tissue bank k= cenobone allograft manufactured by hamanand saz baft kish company ( trc corporation ) g= grafton manufactured by the american tissue bank ( osteotech inc . , eatontown , nj , usa ) e= empty sites without any graft material as control analysis of the data and parameters in the second month i= itb allograft manufactured by the iranian tissue bank k= cenobone allograft manufactured by hamanand saz baft kish company ( trc corporation ) g= grafton manufactured by the american tissue bank ( osteotech inc . , eatontown , nj , usa ) e= empty sites without any graft material as control no foreign body reaction was detected except one in the first month in the control group and one in the second mouth in the grafton group . there were no statistically significant differences in foreign body reaction between the two intervals and among the study groups ( both ps=0.39 , tables 1 and 2 ) . lamellar ( mature ) bone was not observed in any defect in the first interval , and the difference in the regenerated bone type was marginally significant ( p= 0.06 ) . in the second interval , only one of the defects filled with itb exclusively showed lamellar bone and in all other defects , the regenerated bone was mixed ( a mixture of mature and immature bone ) . in the second interval , the four defect types did not exhibit any significant differences in the quality of regenerated bone ( p=0.60 , tables 1 and 2 ) . the mean amount of regenerated bone was 29.46% , 3.55% and 0.93% in defects filled with grafton , itb and cenobone , respectively in the first interval , and 35.23% , 25.29% and 25.36% , respectively , in the second interval . in all defects filled with grafton , bone regeneration was observed in both intervals ( table 3 ) . comparison of the mean bone regeneration by time and materials i= itb allograft manufactured by the iranian tissue bank k= cenobone allograft manufactured by hamanand saz baft kish company ( trc corporation ) g= grafton manufactured by the american tissue bank ( osteotech inc . , eatontown , nj , usa ) e= empty sites without any graft material as control the difference in the amount of regenerated bone during the first month between cenobone and itb groups was trivial , but the difference in the amount of regenerated bone between the grafton and cenobone groups was partially significant ( p=0.06 ) . at the two - month interval , no significant differences were observed among the groups in relation to bone regeneration ( p=0.62 ) . the mean amount of residual material was measured and reported in square - millimeters ( mm ) . on the whole , the maximum and minimum means of the residual graft material were observed in defects filled with grafton and cenobone , respectively , with no statistically significant difference ( p=0.19 and p=0.20 , respectively for the first and second intervals ) . osseous regeneration is one of the most important therapeutic aims and has widespread application in periodontal and implant treatments . the main goal of the present study was to evaluate the regenerative value of two types of allografts manufactured in iran in comparison with a more commonly used product in critical - size defects in rabbit calvarium . chang et al , in 2011 created four 8-mm defects in the calvaria of rabbits to evaluate the biological effects of calcium phosphate combined with cyanoacrylate . produced four 8-mm defects in the calvaria of rabbits in order to evaluate the efficacy of three different types of allografts . rabbits have been widely used by medical researchers due to their easy use , high metabolic rate and maturation in six months . the rate of bone remodeling in rabbits is three times the rate in humans ; therefore , a 4-week time interval appears to be adequate for evaluation of initial healing responses in bone . the rabbit skull is a favorable bone model to carry out experiments with the use of bone graft materials . in addition , rabbit calvarium is considered an experimental site comparable to the alveolar bone because of its intramembranous osteogenesis process . histological evaluations carried out in the current study showed that in the first interval , the most common inflammation grade was mild ; however , in the second interval , the predominant situation was absence of inflammation . this finding , along with the absence of foreign body reaction in the majority of defects , demonstrates a favorable healing process in the area . none of the defects filled with cenobone exhibited acute inflammation , indicating a high rate of adaptation and minimal provocation of the immune system by this material . in studies in which synthetic materials or xenografts are used , acute inflammation and presence of a dense infiltrate of polymorphonuclears are common occurrences . materials that are resorbed slowly ( such as hydroxyapatite ) lead to a chronic inflammatory reaction and they might even be surrounded by a fibrotic encapsulation . acute inflammation can interfere with the regeneration process and can even result in dehiscence of the margins of the wound , which did not occur in the current study . previous researches showed that placing a demineralized allograft ( dbm ) in non - skeletal areas resulted in the formation of ossicles , a phenomenon that was termed osteoinduction . there are differences between the products of different tissue banks in relation to their capacity to induce bone regeneration . osteoinductivity depends on different factors , such as the age and gender of the donor , form , size and origin of the particles and the technique used in the tissue bank to prepare the allograft . it has been reported that dfdba should be harvested from donors under 50 years of age and the best age is under 30 years old . pinholt and solheim placed dbm in the muscular and subcutaneous tissues of mice and reported that osteoinduction potential increased from birth up to puberty but decreased afterwards in addition , donor - related genetic factors and prolongation of the interval between death and harvest of bone affect the regenerative potential of allografts . the best results are achieved with the use of allografts with a particle size of 250750 m [ 16 , 19 ] . allografts are classified based on their mineral content into mineralized ( freeze - dried bone allografts , fdba ) and decalcified ( dbm or dfdba ) groups . decalcification results in the release of factors such as bmps , which induce the differentiation of undifferentiated mesenchymal cells of the recipient site into osteoprogenitor cells . complete decalcification compromises the physical properties of the allograft , although it increases the amount of bmps available . compared dfdba and fdba for the repair of intraosseous periodontal defects and reported no significant differences between them . simion et al . reported favorable results with the use of dfdba based on the guided bone regeneration ( gbr ) philosophy and use of membranes . analyses carried out in the current study showed that the amount of regenerated bone in the first interval decreased from defects containing grafton to defects containing itb and cenobone , respectively and there were great differences between the minimum and maximum amounts of regenerated bone ( table 3 ) . similarly , in the second interval , the maximum amount of bone regeneration was observed in defects containing grafton ; however , the amount of bone regenerated in areas containing itb and cenobone increased by almost similar amounts . although the amount of regenerated bone in all the defects did not exhibit significant differences at the end of the study , osteogenesis during the first and second months did not show a uniform pattern . in other words , there was a delay in bone healing processes in areas containing cenobone and itb . in the grafton group , the maximum bone regeneration occurred during the first month but in the itb and cenobone groups , maximum bone regeneration occurred after the first month . in other words , with an increase in duration of healing and during the two - month period , bone regeneration in areas containing cenobone and itb increased to the level of grafton ( graphs 1 and 2 ) . major differences in osteogenesis among the different groups during the first month minor differences in osteogenesis among the different groups during the second month in a study by lee et al , the osteogenic effects of cortical allografts were compared with those of mixed cortico - cancellous and dbm allografts using the micro - computed tomography technique . the results of the current study were consistent with those of lee et al , with regard to the chronology of regeneration processes . ziran et al . used grafton with lyophilized cancellous bone allograft chips for orthopedic repair of bone traumatic defects and nonunion areas and concluded that such a composite is a good alternative for autogenous bone but should be used with caution in smokers . used cenobone and dembone allografts for the treatment of dehiscence defects around implants and concluded that application of these two dfdba allografts under the collagen membrane and use of gbr technique did not increase the bone - implant contact . sarkarat et al . carried out a human study and compared two types of dfdba allografts , including cenobone and osseo+ ( imtec corporation ) in extraction tooth sockets and concluded that the mounts of viable , non - viable , trabecular and amorphous bone were the same in both groups after three months and even the number of osteoblasts was significantly higher in cenobone biopsies compared to osseo+ biopsies . therefore , both materials were considered suitable to preserve the alveolar ridge after tooth extraction . al , in 2009 evaluated the cell behavior in response to osteoinductive effect of dbm using immunohistochemistry techniques . also , a type of allograft , referred to as dynagraft ( dbm ) has been reported to induce osteogenesis in defects . kaya et al , in 2009 showed that dbm in the form of particles or putty had a similar effect on hard and soft tissue parameters . the results of a human study by abolfazli et al . showed that the reaction of soft tissue to cenobone allograft was favorable and comparable to that of autogenous grafts . in addition , there were no significant differences in bone regeneration and soft and hard tissue parameters with the use of cenobone and autografts in the periodontal defects . in general , since dbm can cause ectopic osteogenesis in muscle and subcutaneous tissues , it is considered osteoinductive . also , dbms contain bmps and low concentrations of growth factors . growth factors play an important role in induction and regulation of wound healing processes and influence cellular functions such as osteogenicity , mitogenicity , chemotaxis , differentiation and metabolism [ 7,2830 ] . evaluation of residual materials in the defects in different groups in our study showed that a significant portion of allografts was resorbed during the first month . the resorption rate of allografts depends on the cortical bone content , processing technique , the form and size of the particles , porosity of particles , inflammation and various other factors . decalcification process decreases the antigenicity of bone ; therefore , dbm is less immunogenic than mineralized allografts , which might be a reason for a low inflammatory response to itb and cenobone allografts . higher resistance of grafton to resorption and more inflammation around it might be attributed to its higher degree of mineralization compared to cenobone and itb . based on the results of the current study , the highest and lowest percentage of residual materials belonged to grafton and cenobone , respectively , indicating a higher rate of osseous regeneration in defects containing grafton versus the lowest numerical value of regeneration in defects containing cenobone . which exhibited the highest resorption rate . in other words , longer presence of allograft in the defect means longer presence of an osteoconductive scaffold and osteoinductive growth factors and thus , results in higher osteogenesis . itb allograft ranked between the two other allografts in relation to the amount of resorption and bone regeneration . however , the results of the current study showed that there was an inverse relationship between allograft resorption rate and the severity of inflammation . in other words , the minimum inflammation and highest resorption rate , and the maximum inflammation and lowest resorption rate were observed with cenobone and grafton allografts , respectively . it appears that easy and fast resorption of cenobone without severe provocation of the immune system decreased the rate of osteogenesis in early stages of healing ; however , in later stages , regeneration was accelerated and osteogenesis improved by the release of growth factors . however , despite differences in the amount of allograft resorption , which were evaluated in the current study , there were no significant differences among the study groups in the amount of residual allograft materials . histological evaluation showed that osteogenesis normally spread from the peripheral areas toward the center of the defects . in some situations , osseous regeneration began at both the central and peripheral sites of the defects and in some defects a bridge or a lateral connection was observed between the central and peripheral areas . this was consistent with the results of studies by khoshzaban et al , and lee et al . lee et al . demonstrated that in the control defects , which had no graft material , bone regeneration occurred only at the periphery and never extended to the central areas ( fig . , in most areas , the regenerated bone was of the immature or woven type and only in limited areas of the defects lamellar bone was seen ( fig . no mature bone was found in the one - month samples ; however , mature bone was observed in all the samples in the second interval . comparison of the type of regenerated bone among the groups showed that bone maturation rate in samples of itb was higher than that in other groups . it appears that longer evaluation intervals resulted in more bone maturation , and higher percentages of lamellar bone formation in all defects . except for the control defects , in some cases , disturbances in bone regeneration were observed in defects containing biomaterials , which might be attributed to the collapse of the periosteum or the dermal flap toward the defect duo to an increase in intracranial pressure and advancement of meningeal membrane into the defects . the main disadvantages of allografts include transmission of human immunodeficiency virus and hepatitis b and c viruses and creutzfeldt - jacob disease . the prevalence of transmission of viruses by allografts was reported to be 1 in 1.5 millions in 1990 . however , it decreased to 1 in 8 millions by drying and freezing the allografts . advances in molecular technology for the control of diseases have significantly decreased the risk of transmission of viral diseases . compared the effects of different techniques for sterilization of allografts on the inductive properties of dbm and concluded that there were no significant differences between radiation with gamma rays and use of ethanol oxide and ethanol ; however , autoclaving decreased the inductive potential of allografts . under the limitations of the present study , the results showed that the allografts evaluated are favorable materials for osseous regeneration and have the potential to regenerate osseous defects in vivo . although there were differences in the amount of bone regeneration in the samples , the differences were not significant . in other words , the results did not indicate significant differences in the amount of bone regeneration among the study groups . therefore , the allografts manufactured in iran , which were evaluated in the current study , can be considered as safe and efficient alternatives for bone grafts , with properties comparable to those of grafton . further animal and human studies are necessary with larger sample sizes and longer follow - ups so that other aspects of the effects of these allografts on the regeneration of bone can be elucidated .
abstractdeep sternal wound infection is a severe complication after open heart surgery . according to the different severity and dimensions of the deep sternal wound infection , the treatment method is different . in this study , we aimed to describe our experience with the rectus abdominis myocutaneous flap for large sternal wound management , especially when 1 or 2 internal mammary arteries were absent.between october 2010 and january 2016 , a retrospective review of 9 patients who suffered from the extensive thoracic defects after deep sternal wound infection was conducted . all of these sternal defects encompassed almost the full length of the sternum after debridement . defect reconstruction was achieved by covering with a rectus abdominis myocutaneous flap . when the ipsilateral or bilateral internal mammary artery had been harvested previously , we took advantage of the inferior epigastric artery to provide additional blood supply to the rectus abdominis myocutaneous flap . thus , this flap had a double blood supply.there was no recurrent infection in all 9 patients . three patients received the rectus abdominis myocutaneous flap with a double blood supply . flap complications occurred in 2 patients ( 22% ) . one patient who did not have the double blood supply flap suffered from necrosis on the distal part of the flap , which was then debrided and reconstructed with a split - skin graft . the other patient had a seroma at the abdomen donor site and was managed conservatively . none of the patients died during the hospital stay.this study suggests that the rectus abdominis myocutaneous flap may be a good choice to repair the entire length of sternal wound . when 1 or 2 internal mammary arteries have been harvested , the inferior epigastric artery can be anastomosed to the second intercostal artery or the internal mammary artery perforator to provide the rectus abdominis myocutaneous flap with a double blood supply .	deep sternal wound infection ( dswi ) is a rare but serious and potentially fatal complication after median sternotomy for cardiac surgery , with an incidence rate 0.5% to 4% and an associated mortality rate between 14% and 47% . presently , management of this problem is still controversial . some reports emphasize that more efforts should be taken to prevent it rather than to treat it , especially for those patients who have high risk factors such as obesity , comorbidities of diabetes mellitus , or chronic obstructive pulmonary disease . however , the introduction of various flaps has led to a significant decrease in morbidity and mortality over the past 30 years . according to the severity and dimensions of the dswi , the therapeutic methods range from conservative treatment to radical surgical debridement followed by negative pressure wound therapy or 1-stage flap coverage . current sternal wound management involves debridement of any devitalized infected soft tissues and bones , administration of culture - specific antibiotics , and the use of a vascularized flap to fill the wound cavity . at present , the pectoralis major muscle or myocutaneous flap remains the most common method for closing the majority of the infected median sternotomy wound . however , this flap does not have the capacity to resurface the entire length of the sternal defect when the thoracic defects are larger than half of the sternum . the greater omentum has the ability to cover the entire wound and control the sternal infection , but perhaps , leads to the spread of the infection to the abdomen . the rectus abdominis myocutaneous flap ( ramf ) has the major advantages of easy dissection , proximity to the infected sternal wound , and provision of ample skin island to repair the extensive thoracic skin deficit . however , several authors reported that this flap may not be considered as a viable option for sternal defects reconstruction when the ipsilateral or bilateral internal mammary artery ( i m a ) had been harvested for coronary artery bypass grafting ( cabg ) . when the ramf is rotated by 180 into the thoracic defects , the inferior epigastric artery ( iea ) can be anastomosed to the intercostal artery or the i m a perforator . thus , this flap possesses a double blood supply . in this study , we aimed to describe our experience with the ramf for large sternal wound management , especially in the absence of 1 or 2 internal mammary arteries . to our knowledge , this is the first introduction of the particular ramf with a double blood supply . between october 2010 and january 2016 , 9 consecutive patients who had received sternal reconstruction with the ramf after radical surgical debridement were referred . the general data ( table 1 ) was collected by reviewing the medical records of the patients . according to the american center of disease control norm , dswi was defined as clinical infection requiring surgical debridement up to and including the sternum , with or without positive microbiology . the 9 patients in this study included 7 males and 2 females , with a mean age of 59 years ( range : 4865 years ) . patients weights ranged from 43 to 95 kg , with a mean weight of 78 kg . all selected patients were received at least 2 debridements ( range : 24 times ) before sternal defect closure , and did not undergo any other flap procedures to repair the defects before the ramf procedure which was performed by the same surgeon ( zhao zheng ) . the second intercostal artery or the second i m a perforator was identified with the handheld doppler probe preoperatively and marked ( to decide which artery was anastomosed to the iea ) . next , the superior epigastric artery ( sea ) and the iea were marked , then the sea iea axis after microbiological specimens were collected , all infected necrotic tissues and bones were removed until healthy solid bone with bleeding margins was found . we carefully protected the internal mammary vessels during resection of the infected bones and cartilaginous structures . the dimensions of the thoracic defects were templated made and marked on the abdominal skin and off the midline . , the inferior epigastric vessels were viewed as a pedicle left at the inferior axis of the flap and the pedicle length was about 8 to 10 cm . then , the flap was rotated by 180 and filled into the sternal defects over suction drains . the abdominal donor defects were repaired by suturing the margin of the remaining anterior rectus sheath to its lateral component and then the skin was closed by relaxation suture . the iea was anastomosed to the second intercostal artery or the second i m a perforator . at the same time , the inferior epigastric vein was anastomosed to the concomitant vein . therefore , the ramf had a double blood supply ( the anastomotic artery and the sea ) . the iea anastomosis was selected according to the following indication : the ipsilateral or bilateral i m a had been harvested previously or the contralateral i m a was ligated during previous cabg and the wound almost encompassed the full length of the sternum . after the operation , between october 2010 and january 2016 , 9 consecutive patients who had received sternal reconstruction with the ramf after radical surgical debridement were referred . the general data ( table 1 ) was collected by reviewing the medical records of the patients . according to the american center of disease control norm , dswi was defined as clinical infection requiring surgical debridement up to and including the sternum , with or without positive microbiology . the 9 patients in this study included 7 males and 2 females , with a mean age of 59 years ( range : 4865 years ) . patients weights ranged from 43 to 95 kg , with a mean weight of 78 kg . all selected patients were received at least 2 debridements ( range : 24 times ) before sternal defect closure , and did not undergo any other flap procedures to repair the defects before the ramf procedure which was performed by the same surgeon ( zhao zheng ) . all procedures were performed under general anesthesia . the second intercostal artery or the second i m a perforator was identified with the handheld doppler probe preoperatively and marked ( to decide which artery was anastomosed to the iea ) . next , the superior epigastric artery ( sea ) and the iea were marked , then the sea after microbiological specimens were collected , all infected necrotic tissues and bones were removed until healthy solid bone with bleeding margins was found . we carefully protected the internal mammary vessels during resection of the infected bones and cartilaginous structures . the dimensions of the thoracic defects were templated made and marked on the abdominal skin and off the midline . , the inferior epigastric vessels were viewed as a pedicle left at the inferior axis of the flap and the pedicle length was about 8 to 10 cm . then , the flap was rotated by 180 and filled into the sternal defects over suction drains . the abdominal donor defects were repaired by suturing the margin of the remaining anterior rectus sheath to its lateral component and then the skin was closed by relaxation suture . the iea was anastomosed to the second intercostal artery or the second i m a perforator . at the same time , the inferior epigastric vein was anastomosed to the concomitant vein . therefore , the ramf had a double blood supply ( the anastomotic artery and the sea ) . the iea anastomosis was selected according to the following indication : the ipsilateral or bilateral i m a had been harvested previously or the contralateral i m a was ligated during previous cabg and the wound almost encompassed the full length of the sternum . after the operation , nine patients who had the extensive thoracic defects were treated by the plastic surgery team during the study period . overall preoperative status six patients were in asa grade 3 and 3 patients in asa grade 4 ( table 1 ) . of the 8 patients who received the cabg , 7 patients had been harvested of 1 or 2 internal mammary arteries and 1 patient had aorto - coronary arterial saphenous vein bypass grafting ( table 1 ) . the mean operation time was 176 minutes ( range : 155228 minutes ) including the vascular anastomosis and the debridement . there was no recurrent infection after the thoracic defects were covered with the ramf combined with antibiotic therapy . one patient who did not have the double blood supply flap suffered from a distal part necrosis of the flap 7 days later and was taken back to the operation room . then , the distal 10% of the flap was debrided and reconstructed with a split - skin graft . the other patient had a seroma at the abdomen donor site and was managed conservatively . the mean overall hospitalization time was 32 days ( range : 2356 days ) after referral to the plastic surgery department . the mean follow - up time was 6 months ( range : 312 months ) . the schematic diagram of the double blood supply ramf for coverage of the entire sternum is shown in fig . a 60-year - old man received coronary artery bypass grafting and his bilateral internal mammary artery had been harvested . after this operation , deep sternal wound infection occurred and 2 debridements were performed by cardiac surgeons . because of the large defect area ( 25 cm10 cm ) , the patient was recommended into our department . defect reconstruction was achieved by rectus abdominis myocutaneous flap combined with the inferior epigastric artery anastomosed to the second intercostal artery perforator . ( a ) large sternal defect after 2 debridements by the cardiac surgeon . ( b ) mediastinal organs were exposed after radical surgical debridement ( c ) defect reconstruction with a double blood supply rectus abdominis myocutaneous flap ( the inferior epigastric artery was anastomosed to the second intercostal artery perforator ) . ( d ) at a 6-month follow - up , no wound infection was found and the patient was satisfied with his own state . the schematic diagram shows the double blood supply rectus abdominis myocutaneous flap for coverage of the entire sternum . ( a ) the red arrow indicates the inferior epigastric vessels , which were left at the inferior axis of the rectus abdominis myocutaneous flap . ( b ) when the rectus abdominis myocutaneous flap was rotated by 180 into the sternal defects , the inferior epigastric artery ( the red arrow ) was anastomosed to the second intercostal artery . at the same time , the inferior epigastric vein was anastomosed to the concomitant vein . treatment of the dswi has improved significantly over the past half century . during the 1960s , the infected sternal wound was managed by local debridement with closed catheter antibiotic irrigation , which led to mortality rate up to 20% . in 1976 , the greater omentum was first reported to cover the extensive thoracic defects after extensive bone and cartilage debridement . later , various muscle or myocutaneous flaps became the primary treatment for reconstruction of the sternal defects . in the 1990s , vacuum - assisted closure dressings occurred and was readily viewed as an adjunct to facilitate sternal wound healing , even as the sole method for definitive treatment in specific cases . today , repairing the wound with well - vascularized muscle or myocutaneous flap after the early radical surgical debridement becomes an effective method . in this study , we used the ramf to repair the extensive thoracic defects containing almost the entire sternum , especially in those patients whose ipsilateral or bilateral i m a had been divided for reasons such as cabg . the rectus abdominis received dual - dominant blood supply from the superior and inferior epigastric arteries , which consisted of a complex perfusion pattern . the sea is a continuation of the i m a and the iea comes from the external iliac artery . with the superior epigastric vessels as the sole blood supply , the rectus abdominis with or without the overlying skin can be raised as a local flap to repair the sternal wound from the manubrium sterni to the xiphoid . the most common complication is the distal part necrosis of the flap because the distal flap can not receive enough blood supply from the pedicle . however , this complication did not occur when the iea was anastomosed to the second intercostal artery or the second i m a perforator in our case series . several studies reported that prior harvesting of ipsilateral or bilateral i m a may preclude the use of the ramf . indeed , we can speculate that the collateral circulation will be strengthened from the anterior intercostal arteries when the i m a has been ligated previously . furthermore , the i m a divides distally behind the sixth or seventh interspace into the sea and the musculophrenic artery . if the i m a is ligated on the distal bifurcation point , the musculophrenic artery will provide a rich supply of collateral circulation to the sea . based on this , some previous studies reported that the rectus abdominis muscle or the myocutaneous flap was used successfully for median sternotomy wounds after ipsilateral i m a ligation . however , this flap is limited to repair the lower half of small sternal wound , which requires a pectoralis major muscle flap for the superior part of the wound . in our study , the entire sternal wound was repaired successfully when we took advantage of the iea to provide additional blood supply to the ramf . there are also other alternative methods to reconstruct the extensive thoracic defects which comprise almost the entire sternum and the significant skin defect after radical surgical debridement . for example , pectoralis major muscle advancement for upper sternum with rectus abdominis muscle turn - over for the coverage of lower sternum is another choice for the entire sternal wound reconstruction . however , we rarely consider the combined use of 2 or more local flaps in our study when a single local flap is sufficient to repair the defect . the greater omentum flap is also a useful reconstructive option because it contains rich vascular supply and large amount of available soft tissues which can reach the entire sternal defects and fill irregular defects sufficiently . the emergence of laparoscopic omental harvest has gained great attention on omental flap use in extensive thoracic defects after dswi . in addition , several previous literatures suggested that the use of omentum may be associated with lower mortality and fewer complications when compared with the pectoralis muscle flap . . moreover , omentum may not be available in those patients with intra - abdominal adhesion after previous surgery . lastly , secondary split - thickness skin graft is necessary when compared with the ramf . more recently , the free myocutaneous flap has been described for sternal wound closure and viewed as a new choice to repair the extensive thoracic defects after dswi . in our case the complication rate of 22% ( 2/9 ) is comparable to that found in previous studies of 25% to 30% . the tip necrosis is the major complication associated with the ramf , which has been reported in a lot of literatures . but this complication did not occur in our patients whose iea had been anastomosed to the second intercostal artery or the i m a perforator . furthermore , even when the ipsilateral or bilateral i m a has been harvested , the particular ramf also has enough blood supply and heals well . despite the satisfactory results that all patients were healed and did not have recurrent infection first , because of the small number of patients included in the study , we can not have enough data for further analysis and make a stronger conclusion . in fact , the small number of eligible patients is largely due to the rare prevalence of the extensive thoracic defects after dswi . in addition , when the bilateral i m a and 1-sided iea are harvested , it may decrease the abdominal wall perfusion and increase the risk of abdominal wall herniation . in conclusion , our study suggests that the ramf may be suitable to repair the large sternal wound up to the entire sternum . when one or both internal mammary arteries have been ligated for cabg or other reasons , the iea can be anastomosed to the second intercostal artery or the i m a perforator to provide the flap with a double blood supply .
miner is web - based software for phylogenetic motif ( pm ) identification . pms are sequence regions ( fragments ) that conserve the overall familial phylogeny . pms have been shown to correspond to a wide variety of catalytic regions , substrate - binding sites and protein interfaces , making them ideal functional site predictions . the miner output provides an intuitive interface for interactive pm sequence analysis and structural visualization . the web implementation of miner is freely available at . source code is available to the academic community on request .	because of the exponential growth of available sequence data , the development of accurate computational strategies for functional site identification has become one of the most important post - genomic challenges ( 1 ) . although attractive owing to their relative simplicity , conservation - based approaches frequently result in too many false positives to be satisfactory . in addition , sequence regions with significant variability can also be critical to function , especially when their composition may define subfamily specificity . frequently , these regions correspond to residues that are critical in molecular recognition and binding specificity . in this report pms are sequence alignment regions that conserve the overall phylogeny of the complete family . through comparison with structural and biochemical data , we have shown that pms represent good functional site predictions in a wide variety of protein systems ( 2 ) . our results indicate that , despite little overall proximity in sequence , pms are structurally clustered around key functionality across a wide variety of structural examples . pms correspond to a variety of structural features , including solvent exposed loops , active site clefts and buried regions surrounding prosthetic groups ( figure 1 ) . our results also indicate that pms are generally conserved in sequence , indicating that pms tend to be motifs in the traditional sense . consequently , pm results bridge evolutionary ( 35 ) and traditional motif ( 6,7 ) approaches . in spite of the small alignment window size if sequences are unaligned , miner will align them for the user by using clustalw ( 8) . a sliding sequence window algorithm is used to quantitatively evaluate the phylogenetic similarity between each sequence region and the whole sequence . phylogenetic similarity is based on tree topology , which is calculated using the partition metric algorithm ( 9 ) . overlapping sequence windows scoring past some preset phylogenetic similarity z - score ( psz ) threshold are identified as pms . empirically , we have determined that a window width between 5 and 10 , and a psz threshold between 1.5 and 2.2 ( lower scores indicate greater similarity ) represent ideal default parameters for functional site prediction . . however , the user retains the option to handle gaps as described previously ( 2 ) . subsequently , partition around medoids clustering of the similarity scores assesses those sequence fragments whose annotation remains in doubt . the accuracy of the approach has been confirmed through comparisons with our manual results ( 2,10 ) . a preprint more thoroughly describing the automated algorithm is available at the miner website . with the automated algorithm in hand , we have precomputed all pms for the most recent version of the cog database ( 11 ) . miner is available as standalone ( command - line based ) software and through the web via a user - friendly interface . after the web - based calculation is complete , miner sends an email with a hyperlink directing the user to their results . miner is part of the larger protein motif analysis portal at california state polytechnic university , pomona ( 12 ) . however , we recommend using 25 or more sequences to ensure sufficient evolutionary diversity . with the exception of gaps , all non - alphabetic characters found in the input will be purged . optionally , a protein data bank ( pdb ) structure may be submitted to better highlight pm regions . miner will automatically add the pdb sequence to a dataset of unaligned sequences if it does not exist . however , user - provided alignments must already include the pdb sequence as part of the alignment . there are several default miner options that can be customized before submission ( figure 2 ) . although masking is optional , we find that eliminating these positions significantly increases the quality of functional site predictions , especially in more divergent families . miner also provides three methods for identifying motifs . by default , miner identifies functional sites as described above . alternatively , miner also provides the option to identify traditional motifs using the false positive expectation ( fpe ) of a regular expression or profile . when used in conjunction with the pm results , these alternative approaches often provide synergistic information . in addition , the width of the sliding window can also be modified . by default , the width is set to five alignment positions , which we find it to be ideal for identifying functional sites ( 2 ) . however , large windows are more appropriate when exploiting motif - ness ( e.g. using pms to de - orfan uncharacterized sequences ) . the z - score threshold is automatically determined by default , but can be manually set any value 1 . finally , either jmol ( default ) or chime viewers can be used for interactive structure visualization . the miner output is a framed html file ( figure 3 ) that provides ( i ) phylogenetic similarity versus window number plots , ( ii ) an annotated structure and ( iii ) an annotated msa . pm regions in the pdb structure are annotated by writing the psz to the temperature factor column . furthermore , interactive structural visualization of the identified pms is achieved with the option of using either jmol or chime . each pm within the alignment is hyperlinked such that clicking it will highlight the corresponding structural region . pm sequence logos , generated by weblogo ( 13 ) , are also hyperlinked from the msa . in all cases , the raw data are available for easy export to auxiliary programs . with the masking feature enabled , regions of the msa colored light gray represent alignment positions that have been purged before pm identification . at the miner website , the tutorial guides one through the output of triosephosphate isomerase results , which is the center of discussion in our previous reports ( 2,10 ) . miner utilizes a sliding sequence window algorithm to systematically evaluate all regions of an msa input . phylogenetic similarity is determined by comparing tree topology , which is calculated using the partition metric algorithm consequently resulting in a psz value . the sensitivity in the pm identification is constrained using a psz threshold , which is automatically determined by default . the resulting miner output uses jmol or chime pdb viewers allowing protein structure and corresponding pm regions to be interactively visualized . the four best - scoring ( psz threshold = 1.5 ) pms identified from the myoglobin protein family are mapped onto structure ( pdbid : 1mba ) . the -carbons of the pms are shown as black spheres ; the heme is shown in gray . the sequence window ( which can toggle between aligned and ungapped ) is hyperlinked to the structure viewer and weblogos . the upper - left window can toggle between both pm ( red ) and fpe ( green ) results . in all cases ,
abetalipoproteinemia ( abl ) is a very rare autosomal recessive disorder caused by mutations in the microsomal triglyceride transfer protein gene ( mttp ) . abl is characterized by lack of lipids and apolipoprotein b ( apob ) in plasma , fat malabsorption and various clinical manifestations . we describe a 12-month - old infant boy , born from consanguineous parents and presented with diarrhea , steatorrhea , growth retardation , hypothyroidism , intraventricular brain cyst and kidney stones . the patient was diagnosed to have abl and treated with dietary modification and oral fat - soluble vitamin replacement and followed until he reached 5 years of age .	abetalipoproteinemia ( abl ; omim 200100 ) is a very rare hereditary autosomal recessive metabolic disorder of fat and fat - soluble vitamins absorption , which is caused by microsomal triglyceride transfer protein ( mttp ; omim 157147 ) deficiency and characterized by absence of plasma apolipoprotein b and ( apo b)-containing lipoproteins . mttp is a chaperone protein found in enterocytes and hepatocytes endoplasmic reticulum ( er ) and includes three structural domains ( n - terminal -barrel , -helix and c - terminal ) and three functional domains ( transfer activity , membrane interaction and lipid binding ) . the molecular basis of this disorder is the inheritance of two mutations in mttp gene which is located on chromosome 4q23 and encodes the large subunit of mttp ( a 97-kda protein containing 894 amino acids ) that forms a heterodimer with the endoplasmic reticulum enzyme protein disulfide isomerase ( pdi ) and accelerates the transfer of lipids onto apolipoprotein b , resulting in assembly and secretion of apo b in the formation of vldl and chylomicrons in the liver and intestine respectively . the syndrome was first described by bassen and kornzweig in 1950 . according to the signs and absence of lipoproteins , salt and colleagues named the disorder as abetalipoproteinemia afterwards . children are usually asymptomatic at birth but develop digestive symptoms including diarrhea , steatorrhea and failure to thrive in infancy . subsequently retinopathy , ataxic neuropathy , acanthocytosis and fatty liver may appear in childhood due to fat - soluble vitamin deficiency , especially vitamin e and beta - carotene that are at extremely low levels in patients with abl . herein , we describe the history and clinical features of a patient with abl who was referred to our hospital and followed until the age of five . a 12-months - old male infant was referred to our hospital because of failure to thrive . he was born from consanguineous parents and his birth weight was 3.4 kg and he failed to gain weight appropriately since two months of age . at the first visit his weight was 6 kg and his height and head circumference were 61 and 44 cm , respectively . he also had developmental delay as he held his neck at 6 months and sat at 9 months of age . he had frequent defecations ( 8 - 9 times a day ) that sometimes were steatotic . he had no family history of similar problems . on physical examination only widening of fontanels was observed . laboratory tests showed : cholesterol=43 mg / dl , triglycerides=6 mg / dl , alanine transaminase ( alt)=17 u / l , aspartate transaminase ( ast ) = 60 u / l , total protein=5 g / dl and albumin=3.8 g / dl . stool examination showed fat droplets ( + ) < 40 drop / hpf . thyroid function test showed tsh=6.5 miu / l and t4=135 nmol / l that established diagnosis of subclinical hypothyroidism . abdominal sonography demonstrated normal size of liver and spleen but there were multiple small stones in both kidneys . macroscopic findings in endoscopy included normal mucosa of esophagus and stomach but snow like appearance and pathologic findings were found in bulb of duodenum . crypts were unremarkable and there was mild infiltration of lymphoplasmacells and some eosinophils ( 3 - 16/hpf ) in lamina properia alongside edema ( figure 1 ) . crypts were unremarkable and there was mild infiltration of lymphoplasmacells and some eosinophils ( 3 - 16/hpf ) in lamina properia alongside edema . it would have been beneficial to perform molecular diagnosis but unfortunately the parents did not agree to do genetic study . in the presence of symptoms , the most probable differential diagnosis included cystic fibrosis ( cf ) and pediatric celiac disease , but severe hypocholesterolemia and hypotriglyceridemia suggested other conditions including abl , homozygous familial hypobetalipoproteinemia ( fhbl ) with dominant transmission ( mutations in apob gene ) and chylomicron retention disease ( cmrd ) with recessive transmission ( mutation in sar1b gene ) . normal levels of tissue transglutaminase antibodies and sweet chloride test along with microscopic studies ruled out celiac disease and cf . family lipid profile was useful in delineating between other conditions . while obligate heterozygote parents of homozygous fhbl patients have decreased levels of plasma ldl - cholesterol and apo b , asymptomatic parents of our patient with normal plasma cholesterol levels suggested a recessive disorder and ruled out fhbl . undetectable amounts of plasma apo b and low density lipoproteins ruled out cmrd and confirmed the diagnosis of abl . our patient was treated with caprilon formula , medium - chain triglycerides ( mct ) oil 1cc / kg / day , vitamin a 10000 iu / day , vitamin e 2000 iu / day , vitamin d 400 iu / day , vitamin k 5 mg daily and levothyroxine 50 - 100 mcg / day . kidney stones resolved with medical treatment and intraventricular brain cyst remained at the same size . liver aminotransferases were elevated ( ast 160 and alt 250 u / l ) but reinforcing of accurate fat - limited diet resulted in normal ranges of these enzymes levels during follow - ups . in last visit he was 5 years old and his weight was 18 kg and his height was 105 cm . abl is a very rare recessive metabolic disorder characterized by the absence of apob - containing lipoproteins in plasma . malabsorption of fat and fat - soluble vitamins leads to a variable clinical phenotype that presents in early childhood with steatorrhea and failure to thrive and may include progressive multi - system abnormalities as the patient ages . most patients are diagnosed in the 2nd to 4th decades and few others in 1st and 6th decades . earlier presentation of symptoms may be due to a more severe phenotype and may be more resistant to medical treatment resulting in poor outcomes . on the other hand , later presentation and longer period of untreated disease may be associated with poor outcomes due to consequences of fat - soluble vitamins deficiency . most reported patients with abl had lipid malabsorption , remarkably low serum lipid levels and gastrointestinal manifestation including diarrhea , steatorrhea and oral fat intolerance . retinitis pigmentosa , myopathy and spinocerebellar ataxia have also been reported in most patients due to fat - soluble vitamins deficiency . our patient had signs and symptoms of early onset abl including fat intolerance , diarrhea , steatorrhea , growth retardation , developmental delay , low tg and cholesterol levels . gastrointestinal manifestations including fat intolerance , diarrhea and steatorrhea are consistent features in most reported patients . brain ct scan showed intraventricular cyst which has not been previously reported despite numerous reported neurological involvement . in abl , both central and peripheral nervous systems can be affected and may be the most serious clinical manifestation . the primary pathology is progressive demeylination of the nervous system which is related to abnormal lipid peroxidation of the highly unsaturated phospholipid of myelin , probably due to prolonged vitamin e deficiency . several studies showed that early high dose vitamin e ( 100 iu / kg ) therapy prior to the age of 2 years can prevent progressive neurological dysfunction and diminish neurological sequelae . pigmentory retinal degeneration is progressive and often causes slowly enlarging annular scotomas with macular sparing . patients may have reduced night vision or color vision , early in the course of disease . previous studies showed that high dose vitamin e therapy or combined vitamin e and vitamin a treatment before 2 years of age can prevent the development and progression of retinopathy . the absence of retinitis pigmentosa in our patient must be attributed to the fact that retinopathy may appear at any time during the first 2 decades of life . red cell acanthocytosis is the hematologic manifestation of abl which is reported in several cases but was not seen in our case . nephrolithiasis was present in our patient which was also been reported previously . in patients with abl fat malabsorption causes combination of unabsorbed fatty acids with calcium ions in the intestinal lumen leading to excessive absorption of dietary oxalate and kidney stone formation . management included dietary fat restrictions , fat soluble vitamins supplements , specific formula and polycitrate that resolved the problem in the patient . our patient had subclinical hypothyroidism , the association between abl and subclinical hypothyroidism was previously reported in another case . the hepatic manifestation of our patient was elevated levels of serum transaminases , probably due to hepatosteatosis which resolved with accurate treatment . in conclusion early diagnosis and accurate treatment are necessary to avoid the complications following fat - soluble vitamin deficiencies .
we compared the analgesic efficacy of spinal and general anaesthesia following transurethral procedures . 97 and 47 patients underwent transurethral bladder tumour resection ( tur - b ) and transurethral prostatectomy ( tur - p ) , respectively . postoperative pain was recorded using an 11-point visual analogue scale ( vas ) . vas score was greatest at discharge from recovery room for general anaesthesia ( p = 0.027 ) . the pattern changed significantly at 8 h and 12 h for general anaesthesia 's efficacy ( p = 0.017 and p = 0.007 , resp . ) . a higher vas score was observed in pt2 patients . patients with resected tumour volume > 10 cm3 exhibited a vas score > 3 at 8 h and 24 h ( p = 0.050 , p = 0.036 , resp . ) . multifocality of bladder tumours induced more pain overall . it seems that spinal anaesthesia is more effective during the first 2 postoperative hours , while general prevails at later stages and at larger traumatic surfaces . finally , we incidentally found that tumour stage plays a significant role in postoperative pain , a point that requires further verification .	pain management during transurethral procedures is a major concern . apart from the standard general anaesthesia , nevertheless , the administered type of anaesthesia is ultimately based on the anaesthesiologist 's decision . recently , local anesthesia with infiltration of the bladder wall or periprostatic nerve blockage was reported [ 13 ] . additionally , sedoanalgesia which is the combination of local anaesthesia with sedation or even the use of virtual reality for pain management has been examined [ 4 , 5 ] . the selection of anaesthesia in a transurethral prostatectomy ( tur - p ) or a transurethral bladder tumor resection ( tur - b ) has been investigated meticulously in previous reports [ 68 ] . in general , regional or local anaesthesia demonstrates distinct advantages and disadvantages in terms of postoperative morbidity [ 68 ] . the aim of the present study was to compare general and spinal anaesthesia in terms of postoperative pain mitigation by recording the patient 's pain perception during the critical first 24 postoperative hours and to investigate a potential correlation of clinical and pathological data with the pain induced by the transurethral procedures . distribution of the patients is depicted in the consort flow diagram ( figure 1 ) . patient age and distribution of the population according to stage and grade of the transitional cell carcinomas ( tcc ) are summarized in table 1 . administration of 1 - 2 g / kg of propofol , followed by 1 - 2 mg / kg of fentanyl along with 1 - 2 mg / kg of suxamethonium . administration of 0.5 mg / kg of atracurium ( n2o at 50% o2 ) and inhaled desflurane at mac 1 ( minimum alveolar concentration ) . spinal anaesthesia was administered as a single shot of 2 ml bupicaine and 1 ml lidocaine without adrenaline . patients with bladder or prostatic capsule perforation , which was identified intraoperatively , were excluded from the study . a 22-f , 3-way dufour catheter was placed in all patients , and bladder irrigation was standard . bladder irrigation was stopped after the completion of the 24-hour observation period after verifying the absence of intravesical clotting by manual irrigation and suction . postoperative pain severity was assessed and recorded using an 11-point visual analogue scale ( vas ) . the vas was given to each patient at the time of his / her arrival in the recovery room , which was considered as hour 0 . each patient recorded his / her pain tolerance at postoperative hours ( h ) 0 , 2 , 4 , 8 , 12 , and 24 . no pain scored 0 points , while worst possible pain for the patient was scored with 10 points . score > 3 during the observation period , 500 mg of paracetamol combined with 20 mg of hyoskine n - butylbromide ( buscopan ) were administered . stratification of pain scoring was made using several parameters , such as age , gender , stage , grade , and location of the tcc 's . statistical analysis was performed using the spss 14.0 for windows statistical package ( spss , inc . , clinical and demographical characteristics were compared using the mann - whitney u test for continues variables and the chi - square test for categorical variables . kruskal - wallis test was used to estimate equality of population medians among groups and the mann - whitney u test for comparison between the groups . the spearman correlation coefficient ( when appropriate vas scores , with 95% confidence interval ( ci ) in relation to postoperative time are shown in figure 2 . no statistical difference was detected in the tur - p patients at any postoperative time between the two anaesthetic methods . in the tur - b patients , mean ( 95% ci ) analogue vas score was greatest at 0 h for general versus spinal anaesthesia ( p = 0.027 ) . at 8 h and 12 h , general anaesthesia 's analgesic efficacy was increased significantly ( p = 0.017 and p = 0.007 , resp . ) after adjusting for gender in the tur - b group , male patients under general anaesthesia experienced more pain at 0 h and 2 h [ mean ( 95% ci ) , 1 ( 08 ) versus 0 ( 08 ) and 1 ( 06 ) versus 0 ( 010 ) , respectively ( p = 0.021 and p = 0.032 ) ] . however , in the female patients , vas score was distributed differently , since spinal versus general anaesthesia 's analgesic efficacy was lost at 4 h [ median ( 95% ci ) 3 ( 04 ) versus 0 ( 03 ) ] , 8 h [ 2.5 ( 03 ) versus 0 ( 02 ) ] and 12 h [ 2 ( 02 ) versus 0 ( 0 - 1 ) ] ( p = 0.005 , p = 0.004 , p < 0.001 , resp . ) , suggesting a better analgesic efficacy for general anaesthesia . a separate analysis was performed in male patients comparing tur - p versus tur - b . no difference in vas score was recorded between the two surgical approaches when general anaesthesia was chosen as analgesic method . in the spinal group , however , tur - b patients presented lower mean vas score than tur - p patients at 0 h [ 0 ( 08 ) versus 0.5 ( 05 ) , ( p = 0.007 ) ] . this pattern changed at 8 h [ 2.5 ( 08 ) versus 0.5 ( 02 ) ( p = 0.039 ) ] and at 12 h , [ 2 ( 08 ) versus 0 ( 02 ) ( p = 0.016 ) ] . interestingly , after adjusting for the covariates stage and grade in the tur - b patients , a higher vas score was observed for pt2 patients compared to pta , pt1 ( tnm ) , and patients with negative pathology report ( figure 3 and table 2 ) . stage and tumor grade were highly correlated ( p < 0.001 ) , but grade did not present any statistical significance with vas score at any postoperative hour . resected tumor volume was used as a categorical variable , and patients were divided in those with < 10 cm [ mean volume 3.3 ( 110 ) , ( n = 82 ) ] and > 10 cm mean 28.1 ( 1140 ) ( n = 15 ) ] . the cut - off point of 10 cm was set by the statistical analysis . those with resected tumor volume > 10 cm presented vas score > 2 at 8h and 24 h , which was statistically significant ( p = 0.050 , p = 0.036 , resp . ) ( figure 4 ) . adjusting for the method of anaesthesia , subjects with bladder tumors larger than 10 cm that received general anaesthesia presented vas score <3 at 4 h , ( or = 6 95% ci 1.0135.04 , p = 0.035 ) . however at 24 h , vas > 3 was more common in patients with tumor volume < 10 cm who underwent general anaesthesia ( or = 3.5 95% ci 1.0911.02 , p = 0.008 ) . apart from the 24 h , tumor located in the lateral bladder walls induced more pain than those situated in the trigone . moreover , tumor multifocality had the highest vas scores in all observation periods ( table 3 ) . several papers compare the effect of these methods in terms of peri- and postoperative morbidity ( blood loss , side effects , and possible complications ) . to our knowledge , this is one of the few attempts of comparing the 2 methods by recording the patient 's pain perception and tolerance . firstly , in patients undergoing tur - p , we found that none of the 2 methods of anaesthesia prevailed during the 24-hour observation period . another report by fredman and colleagues advocated general anaesthesia as the method of choice in transurethral procedures . again , patient satisfaction was recorded , but the main limitation was that no adjustment for the type of the transurethral procedure was made . suggested that regional anaesthesia reduces the incidence of catheter - related pain , although being of similar efficacy to oral diazepam and thus is more advantageous than general anaesthesia . on the other hand , differences were recorded in tur - b patients . to be more specific , spinal was more efficient than general anaesthesia in the first 2 h after surgery , while general proved to be better in the later time points . this fact can be explained by the major implications induced in bladder function by regional anesthesia . indeed , catheter - related pain and detrusor muscle spasm elicited by bladder irrigation can be managed efficiently with regional anaesthesia in the first 2 postoperative hours . however , it simultaneously causes a clinically significant disturbance of bladder function due to interruption of the micturition reflex . additionally , it has a greater effect on bladder compliance and lowers the intraabdominal pressure . as a result , painful bladder overdistention or even acute retention might occur after the removal of the catheter , due to the long - lasting recovery of the normal bladder function . based on the clinical experience , the incidence of such an event is rather rare , thus we might implicate the catheter and bladder spasm as the most distressing causes of postoperative pain . however , an interesting analgesic approach could have been the combination of spinal anaesthesia with antimuscarinic pretreatment , such as oxybutnin or tolterodine , or the administration of gabapentin during general anaesthesia , which is shown to significantly reduce catheter - related pain [ 17 , 18 ] . the above urodynamic effects might be interpreted better in the female patients than in men , since in women there are no prostatic symptoms that could bias the bladder pain , and we should also acknowledge the fact that females perceive pain better than men , as recent reports suggest . in the female population of our study , spinal was less effective than general anaesthesia after the first 4 hours , verifying the possible implications of regional anaesthesia in the female bladder . a very surprising observation is exhibited in the statistical analysis , concerning the bladder tumor stage . patients with higher stage experienced a higher level of pain than the ones with localized disease ( pta - pt1 ) . one can speculate that infiltrative disease stimulates more nociceptors ( highly specialized free endings of sensory nerve fibers ) . even though this is a random result , we believe that it is worth mentioning , since it exhibited statistical significance , and it might motivate a better anaesthetic approach of patients that present with pt2 disease cystoscopically . it is obvious that verification is required for the analysis of a large cohort of patients , which should be also stratified by the type of the applied anaesthesia . we also found that the resected bladder tumor volume becomes a significant parameter of pain induction after a tur - b , when more than 10 cm are resected . on the other hand , when we adjusted our analysis to the type of anaesthesia , general anaesthesia seemed to be more efficient at 4 h and 24 h. tumor volume was the only parameter favoring the efficiency of general anaesthesia independently of the postoperative time of observation . the use of vas scale for the documentation of pain measurement introduces a study bias per se , since this is a subjective method . to date , no objective recording of pain perception has been described , even though several pain scales assessing the patient 's agitation level by recording the facial expression , leg movement , and muscle tension , or even the brain electrophysiologic activity through specialized electroencephalograms ( eeg ) are presented as promising . yet , the use of vital signs , such as heart rate and arterial pressure or even pupil reactions could reflect partially the pain status , but they could be affected also by the postsurgical stress , the patient 's comorbidities , or the prescribed medication . nevertheless , clinically significant differences between vas scores might be considered realistic anecdotally , when they exceed 4 - 5 points . thus , in our study , clinical significant differences were observed in different bladder tumor stages and multiple versus single tumors . the dosage of epidural regimens has an effect of approximately one and a half hours . therefore , it could not have altered the vas scoring , since the 0 h point was set at the arrival in the recovery room . the selection of paracetamol and hyoskine n - butylbromide as rescue analgesics in patients , reporting vas > 3 postoperatively , was made due to their immediate action and their short half - lives [ 22 , 23 ] , which could not have affected the perception of pain during the 2-hour and 4-hour intervals of vas recording . the patients were not randomized due to the fact that the selection of analgesia was made by the anaesthesiologists individually for each patient , according to their performance status . another drawback could be the lack of analysis of the time of the operation and the prostatic volume resected . even though the resected adenoma can not be rationally associated with pain induction , since the major causes of tur - p pain are bladder spasm , catheter - related pain , and capsule offense , a future study could incorporate this parameter . as for the time of the operation , we believe that it could not have affected the result , since the scoring started after the completion of the operation , during which pain is managed sufficiently . the analysis of the advantages and disadvantages of regional compared to general anaesthesia is immensely complicated . as can easily be imagined , the secondary effects of both types of anaesthesia are varied , and realistically , they must be considered for each and every patient , due to the unique clinical profiles presented . this study attempted to establish the potency of general and spinal anaesthesia during transurethral procedures by recording the patient 's pain perception . it seems that spinal anaesthesia is more effective during the first 2 postoperative hours , while general prevails at later stages and at larger traumatic surfaces . finally , we incidentally found that tumor stage plays a significant role in postoperative pain , a point that requires further verification . we advocate a closer cooperation of the urologist with the anaesthesiologist in terms of providing more specific information about the patient 's disease and tailoring the type of anaesthesia to the patient 's needs .
a young male presented with incessant narrow qrs tachycardia and left ventricular dysfunction . 24-holter monitoring revealed multiple episodes of sustained and nonsustained episodes of tachycardia with prolonged sinus pauses at termination . the analysis of the electrocardiogram , followed by an invasive electrophysiological study , suggested an unusual mechanism for this tachy - brady syndrome .	an 18-year old male presented with recurrent palpitations associated with occasional presyncope for the preceding 3 months . nonsustained episodes of narrow qrs tachycardia with sinus pauses at termination were noted on the electrocardiogram ( ecg ) ( fig . holter monitoring showed sustained and nonsustained episodes of tachycardia , with a cumulative duration of 14 h in a 24-h recording , and intervening sinus pauses ( fig . 1c ) lasting up to 4.5 s. his left ventricular ejection fraction was 35% at presentation . during electrophysiological study , sinus cycle length was 734 ms with normal atrio - hisian ( ah ) and his - ventricular ( hv ) intervals . the termination and re - initiation of the tachycardia are shown in fig . 2 . the ecg in fig . 1a and b show a narrow qrs long rp tachycardia with negative p waves in inferior leads . the episodes of this nonsustained tachycardia are initiated with sinus beats , and the second qrs complex of the tachyarrhythmia shows right bundle branch block morphology . the morphology and axis of the subsequent p waves ( p ) are different from the sinus beats . occasionally , the isolated sinus beat is followed by a single p wave , without continuing as tachycardia . the differential diagnosis of this long rp tachycardia includes atrial tachycardia ( at ) originating from inferior atrium , av nodal reentrant tachycardia ( avnrt ) of fast - slow sub - form and persistent form of junctional reciprocating tachycardia ( pjrt ) . during at , the p waves of the initial and subsequent beats tend to be similar , unlike what is observed in this case . the fast - slow type of avnrt is usually initiated following ventricular premature beat and the incessant nature is unusual . orthodromic reentrant tachycardia using an accessory pathway ( ap ) with decremental retrograde conduction properties , i.e. , pjrt can be initiated by sinus beat , and this tachyarrhythmia tends to be incessant . termination on either antegrade or retrograde limb can occur in both reentrant mechanisms , but to terminate with a p wave is unusual for at . 2 helps to confirm pjrt as the mechanism in this case . the initial 2 cycles of tachycardia in fig . 1 bracketing at proximal coronary sinus ( cs ) bipole ( cs 7 - 8 ) . the tachycardia terminates following a paced beat from the right ventricular apex , timed 30 ms later than the his signal . the morphology of this paced complex suggests qrs fusion between the paced and tachycardia beat . termination following a paced ventricular beat not conducted to atrium and resulting in qrs fusion suggests orthodromic av reentry as the tachycardia mechanism . the prolonged local ventriculoatrial ( va ) interval of 210 ms even at the site of earliest retrograde atrial activation during the tachycardia suggests slow conduction through the ap . immediately following its termination , the next sinus notably , the first local v v interval at cs 7 - 8 after the re - initiation is 370 ms , as compared to 360 ms during its stable phase . the increment of 10 ms is contributed by a delay in the ah interval by 60 ms ( 40100 ms ) despite the shortening of the conduction time across the ap by 40 ms ( 210170 ms ) . despite a delay in conduction down the his bundle , the anterograde impulse finds the right bundle in refractoriness , and conducts down the left bundle with an hv interval similar to that in other tachycardia beats . this functional block and its mitigation in the subsequent beats follows that the recovery period in the bundle branch is directly related to the previous rr interval . the conduction times through the pathway , as measured by the local va interval in cs 7 - 8 bipole , showed progressive prolongation ( 170 , 190 and 210 ms ) , in response to a shortening of the preceding local v the progressive reduction in the time interval of the retrograde impulses at the ventricular end of the ap during the re - initiation , related to the conduction delay in the av node in the second beat , unmasked the decremental properties of the ap in this case . similarly , the decremental nature of the conduction in the av node accounted for the minimal changes in av interval in the initial cycles during the re - initiation . frequently , the sinus beats were followed by single atrial echo beats , nonconducted down the av node ( fig . however , if this echo beat could find av node during its relative refractory period and conduct with a prolonged ah interval and bundle branch aberrancy , the reentry could be initiated . during the following cycles , the minimal but incremental prolongation in the conduction time through the ap due to its decremental nature helped the retrograde impulse the tachycardia was incessant during the study and could be terminated only with ventricular pacing . the ap was successfully ablated at cs , within 1 cm of its ostium . holter monitoring done 3 months after the study showed no pauses or tachycardia and the ventricular function recovered completely to suggest tachycardiomyopathy at presentation . symptomatic sinus pauses at the termination of the tachycardia episodes , mimicking a tachy - brady syndrome is unusual in pjrt . suppression of the sinus node related to chronic atrial arrhythmias and its reverse remodeling over weeks to months following successful ablation of the culprit arrhythmia has been described , . however , the complete recovery of sinus node function immediately after the ablation makes this mechanism unlikely . a compensatory vagotonia in response to the initial hypotension and resultant sympathetic activity has been proposed as the dominant mechanism in spontaneous and early termination of supraventricular tachyarrhythmias . however , no significant sinus pauses occurred at the termination of tachycardia episodes on pacing protocols or successful ablation . this also would suggest that a common denominator like vagotonia accounted for the spontaneous termination of tachycardia and the sinus pauses followed . like the av node , the ap mediating pjrt is also known to be vagal sensitive . interestingly , the termination with a block on the antegrade limb was more commonly noted in this case presumably related to a higher sensitivity of the av node to vagotonia compared to the ap .
background : while trying to integrate multiple data sets collected by different researchers , we noticed that the sample names were frequently entered inconsistently . most of the variations appeared to involve punctuation , white space , or their absence , at the juncture between alphabetic and numeric portions of the cell line name.results:reasoning that the variant names could be described in terms of mutations or deletions of character strings , we implemented a simple version of the needleman - wunsch global sequence alignment algorithm and applied it to the cell line names . all correct matches were found by this procedure . incorrect matches only occured when a cell line was present in one data set but not in the other . the raw match scores tended to be substantially worse for the incorrect matches.conclusions:a simple application of the needleman - wunsch global sequence alignment algorithm provides a useful first pass at matching sample names from different data sets .	while trying to integrate multiple data sets collected by different researchers , we noticed that the sample names were frequently entered inconsistently . most of the variations appeared to involve punctuation , white space , or their absence , at the juncture between alphabetic and numeric portions of the cell line name . reasoning that the variant names could be described in terms of mutations or deletions of character strings , we implemented a simple version of the needleman - wunsch global sequence alignment algorithm and applied it to the cell line names . incorrect matches only occured when a cell line was present in one data set but not in the other . a simple application of the needleman - wunsch global sequence alignment algorithm provides a useful first pass at matching sample names from different data sets .
angle class iii malocclusion has been a challenge for researchers concerning diagnosis , prognosis and treatment . it has a prevalence of 5% in the brazilian population , and may have a genetic or environmental etiology . this malocclusion can be classified as dentoalveolar , skeletal or functional , which will determine the prognosis . considering these topics , the aim of this study was to describe and discuss a clinical case with functional class iii malocclusion treated by a two - stage approach ( interceptive and corrective ) , with a long - term follow - up . in this case , the patient was treated with a chincup and an eschler arch , used simultaneously during 14 months , followed by corrective orthodontics . it should be noticed that , in this case , initial diagnosis at the centric relation allowed visualizing the anterior teeth in an edge - to - edge relationship , thereby favoring the prognosis . after completion of the treatment , the patient was followed for a 10-year period , and stability was observed . the clinical treatment results showed that it is possible to achieve favorable outcomes with early management in functional class iii malocclusion patients .	angle class iii malocclusion has raised controversies among researchers concerning diagnosis , prognosis , and treatment . it affects 5% of the brazilian population , with a greater incidence in people of asian origin . in terms of etiology , this problem can have either a genetic origin , with a more unfavorable prognosis , or an environmental origin caused by more anterior and inferior tongue positioning , habits , and oral breathing . class iii malocclusion can be classified as dentoalveolar , skeletal , or functional , which will determine the diagnosis and prognosis . ideally , diagnosis of this malocclusion should be made as early as possible , still during deciduous dentition . early recognition of this discrepancy depends on a careful observation of a sequence of facial , occlusal , and cephalometric characteristics . it is known that class iii malocclusion exacerbates during growth , mainly starting at adolescence . therefore , in children , this malocclusion is not totally defined , and the not yet established facial and occlusal features can complicate the diagnosis . the earlier the interceptive phase is initiated , the greater the orthopedic effects will be to the detriment of the unavoidable orthodontic effects . moreover , an early benefit in terms of esthetics for the child implies improved self - esteem , considering the psychological factor . among the approaches for treating class iii malocclusion is the use of orthopedic appliances , such as chincups , facial masks , functional orthopedic appliances of the jaws , preventive orthodontic appliances ( e.g. : eschler arch and porter appliance or " w " arch ) , multibracket fixed appliances , and a combined orthodontic and orthognatic surgery protocol . the correct indication of orthodontic therapy with a chincup depends on a precise diagnosis of class iii malocclusion . it is advisable to initiate the treatment at an early age , using an upward and backward force from 350 g to 500 g. a cephalometric study performed by graber ( 1977 ) showed that the use of a chincup promoted a backward movement of point b , due to a clockwise rotation of the mandible . the length of the mandible also decreased about 1 mm due to the pressure transmitted by the chincup to the condyle , which generated , on the other hand , a delay in vertical growth . in another study , ( 1984 ) evaluated the skeletal changes produced before , during and after chincup therapy . the authors concluded that chincup therapy would be a very useful and efficient method for correcting class iii with mandibular prognathism . ( 1990 ) verified that an early treatment with a chincup produced positive orthopedic effects on the mandible ; however , they did not assure an improvement in the skeletal profile . another possibility of early interception in class iii treatment consists of an orthopedic / orthodontic appliance , the so - called eschler arch . this appliance has a modified labial bow , which will gently touch the lower incisor labial surface , and an acrylic occlusal bite - raising appliance , which affords normal growth of the maxilla , and helps the correction of the negative overjet . should it be necessary to correct the upper incisor inclination , finger springs can be used for their protrusion . considering the aforementioned therapeutic possibilities , the aim of this study was to describe and discuss the treatment of a patient with angle class iii malocclusion , treated according to a two - stage approach ( interceptive and corrective ) , and a long - term follow - up period . a 9-year - old female patient in the mixed dentition stage ( second transitional period ) was referred for treatment with a chief complaint of an anterior crossbite . during the clinical interview , facial evaluation showed lack of development of the middle third , which is an apparently normal feature for asians . intraoral examination revealed a forward shift of the mandible , with a marked mesial molar relationship , and a crossbite of the four permanent incisors , thus characterizing a functional class iii malocclusion ( figure 1 ) . pretreatment extraoral ( a and b ) and intraoral ( c - g ) photographs . initial panoramic x - ray ( h ) and lateral cephalogram ( i ) ( parents signed informed consent authorizing the publication of these pictures ) a panoramic radiograph revealed the presence of all permanent teeth either erupted or in several developing stages . careful evaluation of lateral cephalograms confirmed a class iii malocclusion , with an acute nasolabial angle , and a horizontal growth pattern . following the confirmation of a class iii malocclusion through the cephalometric analysis , clinical differential diagnosis was accomplished by verifying the occlusion pattern at either the intercuspal position ( ip ) or at the centric relation ( cr ) . the patient showed a crossbite at maximal habitual intercuspation with a forward shift of the mandible and , at cr , a retroposition of the mandible with edge - to - edge contact between the upper and lower incisors . this clinical condition confirmed a functional class iii malocclusion , which greatly favors the orthodontic treatment ( figure 2 ) . frontal intraoral view showing the edge - to - edge contact of the incisors - centric relation ( b ) the patient was at a mixed dentition stage , with great potential of growth , so the main goal of the treatment was to correct the anterior crossbite , while also correcting the functional forward deviation of the mandible , and allowing the maxilla to be in a forward position in relation to the mandible , thus affording a normal development . the proposed treatment protocol comprised two stages : the interceptive and the corrective phases . in the first phase , a chincup was used only at night to maintain mandibular retrusion , and the eschler appliance , also known as " progenic appliance " , was used during the day . the eschler appliance is composed of : a ) retention clasps , e.g. adams clasps for molars , and intermolar auxiliary clasps for deciduous teeth and premolars , b ) an eschler labial bow , made in 0.9-mm wire , and adapted at the labial surface of the lower incisors , c ) an occlusal bite - raising appliance in acrylic resin with a thickness of 2 to 3 mm . if necessary , springs and an expansor screw can be added ( figure 3 ) . extraoral ( a ) and intraoral ( b ) photographs ( parents signed informed consent authorizing the publication of this picture ) the chincup was activated by 1/2 inch elastics . these were changed either weekly or whenever necessary , and produced a force of 350 g to 500 g on each side , directed at an angle of 45 in relation to the occlusal plane . activation of the eschler bow was performed by closing the loop sufficiently to make the bow touch the labial surface of the lower incisors , without overpressure , since the 0.9-mm wire exerts intensified force due to its diameter . this appliance was intended to produce an orthopedic forward movement of the maxilla , and an orthodontic lingual movement of the lower incisors . after correcting the crossbite , this was accomplished in order to allow the eruption of the premolars , eliminate the free space caused by the occlusal opening , and avoid a tongue interposition , and a possible posterior open bite . follow - up appointments were scheduled until the complete correction of the anterior crossbite , totalizing a treatment period of 14 months . when the appliances were removed , the patient was seen every six months , up to the complete development of the permanent dentition , characterizing thus the end of the interceptive phase . in this phase , a marked improvement in both facial harmony and occlusion was observed ( figure 4 ) . end of interceptive phase : extraoral ( a and b ) and intraoral ( c - e ) photographs at the end of the interceptive phase , panoramic x - ray ( f ) and lateral cephalogram ( g ) ( parents signed informed consent authorizing the publication of these pictures ) lateral cephalograms ( figure 4 ) of the end of the interceptive phase showed the successful results of an early treatment with an eschler appliance associated with a chincup . a panoramic radiograph showed the end of the mixed dentition , a good root parallelism , and the presence of the third molars ( figure 4 ) . during the development of the occlusion , the patient was concerned about the gradual increase of the diastema between the central incisors . however , this condition was expected , since the growing mandible caused the proclination of the incisors , thereby increasing arch length ( figure 5 ) . two - year follow up after interceptive phase : extraoral ( a ) and intraoral ( b ) photographs presenting the diastema between upper central incisors . lateral cephalogram ( c ) and panoramic x - ray ( d ) ( parents signed informed consent authorizing the publication of this picture ) approximately 2 years after the interceptive phase , and due to the patient 's dissatisfaction with the diastema , the second phase of this protocol was initiated with the installation of a fixed appliance ( figure 6 ) . the corrective phase aimed to close the diastema and perform small corrections , i.e. , axial inclinations . it lasted for about 14 months and the results are shown in figure 7 . intraoral photographs showing the fixed appliances ( a - c ) end of corrective phase : extraoral ( a , b ) and intraoral ( c - e ) photographs , panoramic x - ray ( f ) and lateral cephalogram ( g ) ( parents signed informed consent authorizing the publication of these pictures ) ten years after the corrective treatment , a new follow - up appointment verified stability of the treatment , and the final result can be seen in figure 8 . follow - up at 10 years after the treatment : extraoral ( a and b ) and intraoral ( c - g ) photographs , panoramic x - ray ( h ) and lateral cephalogram ( i ) ( patient signed informed consent authorizing the publication of these pictures ) superposition of initial , final , and follow - up cephalometric tracings of interceptive phase ( a ) . superposition of initial , final , and follow - up cephalometric tracings of corrective phase ( b ) in this case , class iii was intercepted , and a fixed appliance was installed only to correct small rotations , the anterior diastema , and to improve axial teeth relationships . after the cephalometric analysis ( table 1 ) , it was verified that the sna angle continued to increase , while the snb angle and co - gn were unaltered during the interceptive phase . the measurements representing the vertical position of the mandible , fma , and sn.gogn , were stable . the changes in linear and angular measurements of the upper and lower incisors contributed to obtain a positive overjet . from 9 to 12 years of age a proclination of the upper incisors from 20 to 25 , and a retroclination of the lower incisors from 30 to 22 were observed . initial , final , and post - interceptive control cephalometric values corrective orthodontic treatment was initiated 2 years after the finalization of the interceptive phase . table 1 shows the cephalometric values at the initial , final , and 10-year post - corrective follow - up . it was observed that the anb angle remained positive due to the stability of both sna and snb angles , as well as those for fma and sn.gogn . conversely , co - gn showed an increase of 13 mm , from the end of the interceptive phase to the 10-year follow - up after the corrective phase , showing a value similar to normal mandibular growth . the measurements related to incisor inclinations remained stable at the 10-year follow - up , contributing for the maintenance of the positive overjet . the cephalometric analysis of the case under study demonstrated an increase of the anb angle , mandible growth , and mandibular plane stability . the anb was altered to a favorable value in the relationship of the jaws due to the treatment . the linear and angular measurement changes of upper and lower incisors helped to obtain a positive overjet . ( 1984 ) , in a longitudinal study with skeletal class iii patients , radiographically evaluated the skeletal changes before , during and after chincup therapy . these authors demonstrated that the favorable outcomes obtained were limited to the corrective phase , but returned to the initial features , at the post - treatment period . this demonstrates that the prognosis of cases with great skeletal involvement would not be favorable , unlike the cases described in this report . according to previous studies , there should be proportional values between co - a and co - gn . this case initially demonstrated a co - a measurement of 80 mm , which should be 83 mm , suggesting a maxillary deficiency . however , at the last follow - up appointment , this measurement was 88.5 mm , which has been considered appropriate for a co - gn of 114.5 mm . this study demonstrated the achievement of optimal results , and the stability of the correction of a functional class iii malocclusion treated with a progenic appliance associated with a chincup , and followed by corrective orthodontics . in spite of the good outcomes achieved in this case , further long - term clinical investigations are necessary to assure the stability of class iii treatment . this case report shows that that the stability of the correction of a functional class iii malocclusion with minor skeletal involvement is related to both the correct diagnosis and the early intervention . this treatment allowed proper facial growth and development , preventing worsening of the malocclusion , with more severe consequences .
oral ulcers are a common symptom in clinical practice . among various causative factors , different types of ulcers in oral cavity exist . among this , traumatic ulcerative granuloma with stromal eosinophilia ( tugse ) appears to be quite neglected by the clinicians due to the limited knowledge and awareness . on reviewing with a detailed approach to titles and abstracts of articles eliminating duplicates , 40 relevant articles were considered . randomized studies , review articles , case reports and abstracts were included while conference papers and posters were excluded . of importance , tugse cases been reported only to a minimal extent in the literature . lack of its awareness tends to lead clinicians to a misconception of cancer . thus , this particular lesion needs to be differentiated from other malignant lesions to provide a proper mode of treatment . the present article reviews various aspects of the tugse with emphasis on the clinical manifestation , pathogenesis , histological , and immunohistochemical study . this study provides the clinician contemporaries , a humble expansion to their knowledge of the disease , based on the searched literature , enabling a more comprehensive management of this rare occurrence .	on reviewing with a detailed approach to titles and abstracts of articles eliminating duplicates , 40 relevant articles were considered . these articles were found in pubmed , science direct , scopus and google scholar from first reported tugse case till date were evaluated . randomized studies , review articles , case reports and abstracts were included while conference papers and posters were excluded . on reviewing with a detailed approach to titles and abstracts of articles eliminating duplicates , 40 relevant articles were considered . these articles were found in pubmed , science direct , scopus and google scholar from first reported tugse case till date were evaluated . randomized studies , review articles , case reports and abstracts were included while conference papers and posters were excluded . the pathogenesis of tugse still remains controversial . among all etiological factors , mucosal trauma ( external or internal physical , chemical ; thermal or electrical ) appears to be the major instigating factor of this lesion . clinical and histological presentation is , as an ulcerated lesion with a massive immune response . the inflammatory infiltrate in tugse exhibits sheets of atypical mononuclear cells ( predominantly t - cell lymphocyte population ) , eosinophils , macrophages / histiocytes , and few plasma cells . in earlier studies , immunohistochemistry revealed these atypical mononuclear cells to be belonging to the macrophage and myofibroblast lineage . more recent studies point toward the t lymphocytic function of these cells immunohistochemically , with positivity against all t - cell specific antigens . of late , tugse is agreed upon as a reactive lesion dominated by predominant clonal t - cell population . the exact role of these inflammatory cells to produce such an exaggerated inflammatory response mimicking a malignancy is still unknown . correlating various studies , this appears similar to a hypersensitivity type reaction ( predominant t - cell infiltration exhibiting a delayed type of cell - mediated immunity ) . traumatic ulcerative granuloma with stromal eosinophilia is evoked in most cases , by a traumatic antigen , wherein existing macrophages and histiocytes act as antigen presenting cells . various antigens include toxins , viral , microorganisms , endogeneous degradation products or foreign proteins . on release of cytokines , the predominant cells of eosinophils and lymphocytes are recruited to the submucosal tissue . however , previous studies suggest that the exact presence of eosinophils remains controversial , as most of the traumatic ulcers are devoid of eosinophilic infiltration . on the contrary , certain other authors have concluded that there is increased prevalence of histiocytes and eosinophils than lymphocytes suggesting the aggressiveness of tugse . this process of cyclic reaction continues with increase in proliferating t - cells by release of cytokines / toxic products by the degranulating eosinophils . the accumulation of all these inflammatory cells along with the varied spectrum of cytokines results in tissue damage . in addition , studies also depict that a response resulting from an exaggerated mast cell - eosinophil reaction may arise similar to that noticed in the pathogenesis of type 1 hypersensitivity reaction . degranulating mast cells releases several mediators like eosinophil chemotactic factor , aryl sulfates arid histamines , and major basic proteins , eventually causing tissue destruction . what is interesting to the researchers , is the uniqueness of tugse in being a slow self - healing ulcer with atypical mononuclear cells and massive eosinophilia . eosinophils appear to be a reactive tissue response to an unknown antigen entering via mucosal trauma . the delayed healing process may be attributed to the decreased synthesis of transforming growth factor , released by the eosinophils in the inflammatory infiltrate of tugse as compared to the eosinophils in other traumatic ulcers . furthermore , eosinophils produces a spectrum of cytokines , like tumor necrosis factor , which induces further tissue damage . the pathogenesis of tugse still remains controversial . among all etiological factors , mucosal trauma ( external or internal physical , chemical ; thermal or electrical ) appears to be the major instigating factor of this lesion . clinical and histological presentation is , as an ulcerated lesion with a massive immune response . the inflammatory infiltrate in tugse exhibits sheets of atypical mononuclear cells ( predominantly t - cell lymphocyte population ) , eosinophils , macrophages / histiocytes , and few plasma cells . in earlier studies , immunohistochemistry revealed these atypical mononuclear cells to be belonging to the macrophage and myofibroblast lineage . more recent studies point toward the t lymphocytic function of these cells immunohistochemically , with positivity against all t - cell specific antigens . of late , tugse is agreed upon as a reactive lesion dominated by predominant clonal t - cell population . the exact role of these inflammatory cells to produce such an exaggerated inflammatory response mimicking a malignancy is still unknown . correlating various studies , this appears similar to a hypersensitivity type reaction ( predominant t - cell infiltration exhibiting a delayed type of cell - mediated immunity ) . traumatic ulcerative granuloma with stromal eosinophilia is evoked in most cases , by a traumatic antigen , wherein existing macrophages and histiocytes act as antigen presenting cells . various antigens include toxins , viral , microorganisms , endogeneous degradation products or foreign proteins . on release of cytokines , the predominant cells of eosinophils and lymphocytes are recruited to the submucosal tissue . however , previous studies suggest that the exact presence of eosinophils remains controversial , as most of the traumatic ulcers are devoid of eosinophilic infiltration . on the contrary , certain other authors have concluded that there is increased prevalence of histiocytes and eosinophils than lymphocytes suggesting the aggressiveness of tugse . this process of cyclic reaction continues with increase in proliferating t - cells by release of cytokines / toxic products by the degranulating eosinophils . the accumulation of all these inflammatory cells along with the varied spectrum of cytokines results in tissue damage . in addition , studies also depict that a response resulting from an exaggerated mast cell - eosinophil reaction may arise similar to that noticed in the pathogenesis of type 1 hypersensitivity reaction . degranulating mast cells releases several mediators like eosinophil chemotactic factor , aryl sulfates arid histamines , and major basic proteins , eventually causing tissue destruction . what is interesting to the researchers , is the uniqueness of tugse in being a slow self - healing ulcer with atypical mononuclear cells and massive eosinophilia . eosinophils appear to be a reactive tissue response to an unknown antigen entering via mucosal trauma . the delayed healing process may be attributed to the decreased synthesis of transforming growth factor , released by the eosinophils in the inflammatory infiltrate of tugse as compared to the eosinophils in other traumatic ulcers . furthermore , eosinophils produces a spectrum of cytokines , like tumor necrosis factor , which induces further tissue damage . traumatic ulcerative granuloma with stromal eosinophilia presents as a chronic slow progressive lesion ( commonly fourth to sixth decade ) . predominantly , tugse appears to be more common in males than females ( 1.6:1 ) which may exist from 1-week to several weeks . it appears as a red lesion in oral mucosa with rolled , elevated and indurated margins with central areas of fibrinopurulent membrane mimicking squamous cell carcinoma . even though , tongue is the most common site , other regions like buccal mucosa , vestibular mucosa , gingiva , lip , mucobuccal fold , retromolar area , and palate also may exhibit tugse . histopathologically , tugse exhibits ulceration with diffuse polymorphic inflammatory infiltrate ( exhibiting epitheliotropism ) extending from the superficial epithelium into the deep connective tissue . this dense chronic inflammatory infiltrate includes small round t - lymphocytes , large atypical mononuclear cells , granular macrophages , b - lymphocytes massive eosinophils , plasma cells , and histiocytes in the superficial layer and deep muscle layers . proliferating endothelial cells , blood vessels and focal areas of necrosis are seen scattered throughout the lesion . however , presence of massive eosinophilia is not mandatory in this lesion . a tugse lesion often seen in infants ( riga - fede disease ) occurs within 1-week to 1-year . mucosal lesions are often caused by repetitive traumatic damage due to backward and forward motions of the tongue over the erupting lower incisors , natal or neonatal teeth . rarely , this lesion has also been termed as atypical histiocytic granuloma as studies have shown diffuse scattered atypical large mononuclear cells ( large cells with irregular nuclear contours , fine chromatin , small nucleoli , and abundant cytoplasm ) in a background of diffuse inflammatory cell infiltrate . immunohistochemically , most of these mononuclear cells show cd30 , cd3 and t - cell intracytoplasmic antigen 1 ( t - cell lineage markers ) positivity for t - cells , while few are positive for cd68 macrophages / histiocytic origin and 1050% cells show ki-67 with high degree of production . literature also previously stated that mononuclear cells show factor xiii positivity suggestive of dendritic cells and vimentin positive suggestive of myofibroblasts . regarding the existence of predominantly cd30 + , what remains unresolved is whether this lesion tugse is just a benign oral counterpart of cd30 + lymphoproliferative disease ( lpd ) or a reactive benign primary lesion or just a histological simulator of cd30 + lpd . immunohistochemical studies are slightly in favor of tugse being a benign oral counterpart of primary cutaneous cd30 + lpds . confusion however persists , as cd30 + is expressed on b and t lymphocytes , not only in malignancies , but also in many other lymphoproliferative disorders . the polymerase chain reaction of t - cell receptor gene gamma with monoclonality is suggested for a more definitive diagnosis . differential diagnosis includes primary sypilitic chancre , cd30 + lpd , malignant t lymphoma , angiolymphoid hyperplasia with eosinophilia , langerhans cell histiocytosis , pseudolymphoma , metastatic tumors , kimura 's disease , salivary gland tumors , lymphomatoid papulosis . the mere presence of eosinophils should differentiate from insects / parasites , granuloma faciale , allergic reactions , angiolymphoid hyperplasia with eosinophilic lymphoid granuloma , eosinophilic fascitis , eosinophilic granuloma , and histiocytosis x. traumatic ulcerative granuloma with stromal eosinophilia lesions are treated with antibiotics like penicillin , removal of traumatic agents , followed by excision if still persistent . appropriate management of the offending tooth is suggested in case of riga - fede disease . other modes of treatment include 0.1% triamcinolone acetonide mouthwash , oral antibiotics , steroids ( topical / systemic ) , electrocoagulation , irradiation , and liquid nitrogen . due to the aggressiveness of the lesion , though a few cases show a retarded healing phase , majority of them show a rapid healing following surgery . prognosis of tugse is normally favorable , yet a long term follow up to at least 2 years is mandatory . this condition likely represents a group of related disorders with overlapping clinical and histopathologic features . we , as clinicians , must have adequate knowledge regarding tugse pathogenesis , and perform biopsy for a histological analysis along with a background immunohistochemistry techniques , to rule off other neoplastic lesions at a very early stage .
backgroundassessing therapeutic efficacy and patient satisfaction objectively and quantitatively has always been a problem in patients with vasospastic disorders . we aimed to present the additive value of ultrasonographic assessment of peripheral arteries secondary to cold stimulation , as a test for treatment efficacy during follow-up.material/methodsarterial blood flow rates were measured from radial artery with doppler usg in patients who presented to our department with vasospastic disorders . ultrasonography was performed at the following intervals ; before cold stimulation and at 5th , 10th , 15th , 20th minutes of cold stimulation . patients were controlled by repeat cold stimulation test and doppler us at the 2nd month of the treatment . results were analyzed with spss for mac 20.0 package program.resultswe enrolled 46 patients in the study . all patients were male and mean age was 22.32.17 years . most common symptoms were cyanosis and coldness . there were statistically significant differences between pre - treatment and post - treatment arterial blood flow rates at each measurement time point ( p<0.001 ) except initial measurement ( p>0.05 ) . on post - treatment values , there were 10.040.78 cm / s increase in 5th minute , 6.251.39 cm / s in 10th minute , 6.432.13 cm / s in 15th minute , and 6.381.86 cm / s in 20th minute measurements . all increases at the 5 time points were statistically meaningful when compared to their pre - treatment corresponding time points ( p<0.001).conclusionsdoppler flowmetry added to standard cold stimulation test for evaluating the patients with vasospastic disorders provides better and more objective results when compared to the patient - oriented subjective scoring systems .	vasospastic disorder ( vd ) is a relatively common disorder worldwide population , with a prevalence of 3.3% to 22% . episodic cyanosis , swelling , and pallor on the distal part of the extremities with cold exposure are the main characteristic features of this disorder . this disorder has female dominancy and is especially seen in the 2 and 3 decades of life . if the pathogenesis depends on an underlying disorder , it is called secondary ( obstructive ) form . the remaining form without any underlying disorders is called primary form . since the first description of this disorder , several etiologic factors have been reported to be responsible for the pathogenesis , but it is still not well known , and every possible etiologic factor is still controversial . in addition , no objective , quantitative diagnostic tool or objective follow - up method has been defined . in this study , we aimed to answer the question can doppler ultrasonography be used for objective follow - up of patients with vasospastic disorder ? this is a retrospective study , approved by the local ethics committee of gulhane military academy of medicine . we aimed to describe a quantitative method for the follow - up of patients with vasospastic disorder . between april 2011 and october 2013 , data were collected from files of 46 patients with the diagnosis of vasospastic disorder lasting more than 2 years . the diagnostic criteria , which were defined by wigley et al . , were used as diagnostic criteria for the enrolled patients . several etiologic disorders such as systemic sclerosis and sle , which can cause secondary vasospastic disorder , were excluded , with detailed physical examination and several laboratory findings . demographic data and additional risk factors of patients were recorded . in our department , the cold stimulation test ( cst ) is routinely performed to all patients with vasospastic disorders symptomatology due to our national and military regulations . according to our protocol , application of the cst is as follows : 1 . hand temperatures of the patients are measured with a probe , which is inserted between the pulp of first and second distal phalanges . at the same time , radial arterial blood flow rate is measured at the level of wrist by doppler usg ( detailed below ) . the most affected hand is immersed into the iced - water at 4c for 20 seconds . 3 . after drying the hands , the temperature and the blood flow rate of the radial artery are measured again at the 5 , 10 , 15 and 20 minutes , with the same technique detailed before . the test is performed after resting at room temperature ( approximately 26c ) for at least 30 minutes . patients are classified according to their smoking habits , but a standardized protocol about timing of refraining from tobacco prior to testing was not used . in addition to cst , we routinely use a scale ( verbal complaint severity scale , vcss ) , which is used by our department for evaluating the progress in the follow - up period . in this scale , a physician asks all of the patients about their symptoms for determining their progress . the scale is detailed in table 1 . simultaneously with cst , patients radial arterial blood flow rate was measured at the level of the wrist . for this measurement , radial arterial blood flow velocity was calculated at the level of the wrist on color doppler ultrasonography ( figure 1 ) . logiq book xp scanner ( ge medical systems , usa ) is used as the doppler usg device . in our clinic this combination includes pentoxifylline 1200 mg / d bid ( 2600 mg ) as a vasodilator molecule , nifedipine 60 mg / d bid ( 230 mg ) as a calcium - channel blocker , and acetylsalicylic acid 300 mg / d ( 1300 mg ) as an antiplatelet agent . after 2 months , we evaluate all patients again to observe their progress . after the second evaluation , patients who do not respond to treatment are referred to interventional treatment methods , or they are not allowed to continue their military duties . demographic data , co - morbid disorders , vcss , and blood flow rates of the included patients at pre - treatment and post - treatment period were all recorded . spss for mac 20.0 package program ( spss inc , chicago , il ) was used for statistical evaluation . descriptive results are expressed as mean standard deviation for normally distributed continuous variables and median values for abnormally distributed continuous variables . comparisons of the parametric values were performed with student t test for normally distributed groups and with mann - whitney u test and wilcoxon signed ranks test with abnormally distributed groups . pearson s chi - square test , fisher s exact test , and mcnemar - bowker test were used for the comparisons of categorical variables . a p value of < 0.05 was considered as statistically significant with a 95% confidence interval . between april 2011 and october 2013 , data were collected from files of 46 patients with the diagnosis of vasospastic disorder lasting more than 2 years . the diagnostic criteria , which were defined by wigley et al . , were used as diagnostic criteria for the enrolled patients . several etiologic disorders such as systemic sclerosis and sle , which can cause secondary vasospastic disorder , were excluded , with detailed physical examination and several laboratory findings . in our department , the cold stimulation test ( cst ) is routinely performed to all patients with vasospastic disorders symptomatology due to our national and military regulations . hand temperatures of the patients are measured with a probe , which is inserted between the pulp of first and second distal phalanges . at the same time , radial arterial blood flow rate is measured at the level of wrist by doppler usg ( detailed below ) . the most affected hand is immersed into the iced - water at 4c for 20 seconds . 3 . after drying the hands , the temperature and the blood flow rate of the radial artery are measured again at the 5 , 10 , 15 and 20 minutes , with the same technique detailed before . the test is performed after resting at room temperature ( approximately 26c ) for at least 30 minutes . patients are classified according to their smoking habits , but a standardized protocol about timing of refraining from tobacco prior to testing was not used . in addition to cst , we routinely use a scale ( verbal complaint severity scale , vcss ) , which is used by our department for evaluating the progress in the follow - up period . in this scale , a physician asks all of the patients about their symptoms for determining their progress . simultaneously with cst , patients radial arterial blood flow rate was measured at the level of the wrist . for this measurement , radial arterial blood flow velocity was calculated at the level of the wrist on color doppler ultrasonography ( figure 1 ) . logiq book xp scanner ( ge medical systems , usa ) is used as the doppler usg device . in our clinic , we routinely use a combination therapy for vasospastic disorders for a 2-month period . this combination includes pentoxifylline 1200 mg / d bid ( 2600 mg ) as a vasodilator molecule , nifedipine 60 mg / d bid ( 230 mg ) as a calcium - channel blocker , and acetylsalicylic acid 300 mg / d ( 1300 mg ) as an antiplatelet agent . after 2 months , we evaluate all patients again to observe their progress . after the second evaluation , patients who do not respond to treatment are referred to interventional treatment methods , or they are not allowed to continue their military duties . demographic data , co - morbid disorders , vcss , and blood flow rates of the included patients at pre - treatment and post - treatment period were all recorded . spss for mac 20.0 package program ( spss inc , chicago , il ) was used for statistical evaluation . descriptive results are expressed as mean standard deviation for normally distributed continuous variables and median values for abnormally distributed continuous variables . comparisons of the parametric values were performed with student t test for normally distributed groups and with mann - whitney u test and wilcoxon signed ranks test with abnormally distributed groups . pearson s chi - square test , fisher s exact test , and mcnemar - bowker test were used for the comparisons of categorical variables . a p value of < 0.05 was considered as statistically significant with a 95% confidence interval . in turkey , military service is mandatory for every male aged 20 years . therefore , all the study population was young males ; 32 of the patients ( 69.5% ) were smokers and 14 of these 32 patients were smokers for more than 10 years . none of the patients had co - morbid disorders such as hypertension or diabetes mellitus . the most common complaints were cyanosis ( 34 patients , 73.9% ) , numbness ( 30 patients , 65.2% ) , and hyperhidrosis ( 29 patients , 63% ) . there was a significant decrease in arterial blood flow after cold exposure in both pre - treatment and post - treatment measurements . in the rewarming period , flow rate did not reach its basal values over the test time ( 20 minutes ) in pre - treatment measurements , but it reached its basal value by approximately 10 minute in post - treatment measurements . the changes of arterial blood flow rates during the cst are given in figure 2 . there were statistically significant differences between pre - treatment and post - treatment arterial blood flow rates at each measurement time points ( p<0.001 ) except initial measurement ( p>0.05 ) . on post - treatment values , there were 10.040.78 cm / s increase in the 5 minute , 6.251.39 cm / s in 10 minute , 6.432.13 cm / s in 15 minute , and 6.381.86 cm / s in 20 minute measurements . all increases at the 5 time points were statistically meaningful when compared to their pre - treatment corresponding time points ( p<0.001 ) . in turkey , military service is mandatory for every male aged 20 years . therefore , all the study population was young males ; 32 of the patients ( 69.5% ) were smokers and 14 of these 32 patients were smokers for more than 10 years . none of the patients had co - morbid disorders such as hypertension or diabetes mellitus . the most common complaints were cyanosis ( 34 patients , 73.9% ) , numbness ( 30 patients , 65.2% ) , and hyperhidrosis ( 29 patients , 63% ) . patients were categorized into 3 groups according to vcss . there were no statistically significant differences in patient vcss distribution . there was a significant decrease in arterial blood flow after cold exposure in both pre - treatment and post - treatment measurements . in the rewarming period , flow rate did not reach its basal values over the test time ( 20 minutes ) in pre - treatment measurements , but it reached its basal value by approximately 10 minute in post - treatment measurements . the changes of arterial blood flow rates during the cst are given in figure 2 . there were statistically significant differences between pre - treatment and post - treatment arterial blood flow rates at each measurement time points ( p<0.001 ) except initial measurement ( p>0.05 ) . on post - treatment values , there were 10.040.78 cm / s increase in the 5 minute , 6.251.39 cm / s in 10 minute , 6.432.13 cm / s in 15 minute , and 6.381.86 cm / s in 20 minute measurements . all increases at the 5 time points were statistically meaningful when compared to their pre - treatment corresponding time points ( p<0.001 ) . several diagnostic methods and invasive and non - invasive techniques were defined for the diagnosis of vasospastic disorders over time . however , no definitive diagnostic tool or objective and quantitative follow - up method has been defined . the main cause of this failure is the complexity of the etiopathogenesis , which is still unclear . most patients usually complain of many subjective symptoms such as cyanosis , numbness , or painful attacks , which are often aggravated by cold exposure . therefore , objective diagnosis of primary vasospastic disorders is often not possible . to address this diagnostic problem , several tools ( e.g. , triphasic color changes of hands , finger systolic blood pressure , finger - brachial index , plethysmography , capillaroscopy , and rarely , more invasive techniques ) were used in the history of treating this disorder . unfortunately , most of these diagnostic tools require special conditions special technicians , devices , and sometimes laboratory conditions that can not be used in outpatient conditions . it is also too hard to use these techniques in follow - up , even if they could be used for diagnosis . however , for evaluating patient progress , objective , quantitative , and easily performed tests are necessary . cst and its application method are still not standardized worldwide and there are few studies about this method in the literature . we have used our clinical protocol , as detailed above , for approximately 3 years . used 5c water for cold stimulation in their study and they declared that this temperature is better for provoking the vasospasm than water at 10c . therefore , we routinely used 4c ice - water for provoking the vasospasm , as in our present study . in the literature there are few studies about cold stimulation and its effect on ultrasonographic arterial blood flow rates . most of these studies are focused on the differential diagnosis of the primary form vs. secondary form by using doppler ultrasonography [ 911 ] . there have been no studies on its usefulness as a marker of treatment efficacy in the follow - up period . we consider that our present study is the first in this area . when we analyzed the ultrasonographic parameters of our study , we observed different patterns of blood flow rates . in pre - treatment measurements , flow rates were not able to reach their basal value throughout the test , which suggests that vasospasm lasts longer . in contrast , in the post - treatment tests , flow rates increased quickly to their baseline values by approximately just 10 minutes . in addition , different from the pre - treatment test , flow rates were above baseline values at the 15 minute measurements , which suggests that vasospasm lasts less than the 15 minutes . in our department , we use the verbal complaint severity scale ( vcss ) for classification of disease severity . in our study population , despite the given treatment modality , there were no statistically significant changes in patient vcss . when we analyzed these 3 patients and compared their cst results independent from the study group , we found that there were no statistically significant differences between ultrasonographic flow rates before and after treatment ( p>0.05 ) . in this regard , ultrasonographic flow rate measurements added to cst in our study seem to be helpful for follow - up of patients with vd , both quantitatively and objectively . doppler flowmetry added to standard cold stimulation test for evaluating patients with vasospastic disorders provides better and more objective results when compared to patient - oriented subjective scoring systems . this study was performed in a military hospital ; therefore , all of the patients were male . the smoking time , smoking cessation time , or the correlation between smoking habits and study protocol were not detailed in this study . the significant cut - off value of ultrasonographic blood flow rate changes ( positive or negative ) is not well known and was not defined in this study . to define clear - cut values of this parameter this study was performed in a military hospital ; therefore , all of the patients were male . the smoking time , smoking cessation time , or the correlation between smoking habits and study protocol were not detailed in this study . the significant cut - off value of ultrasonographic blood flow rate changes ( positive or negative ) is not well known and was not defined in this study . to define clear - cut values of this parameter
purposeto report outcomes of exogenous fungal endophthalmitis treated with combination of intravitreal antifungal agents.designretrospective , non - randomized , interventional , consecutive case series.methodstwelve eyes of twelve consecutive cases of filamentous fungal endophthalmitis were treated with a combination of intravitreal amphotericin - b and intravitreal voriconazole ( amb - vo regime ) along with pars plana vitrectomy at a single center . clinical characteristics , microbiology results , treatment strategy , visual , and anatomical outcomes were analyzed.resultsten cases out of the twelve were postoperative endophthalmitis of which nine were part of a post cataract surgery cluster . the remaining included endophthalmitis following keratitis post pterygium excision ( 1 ) and following open globe injury ( 2 ) . the most common fungus was aspergillus terreus , which was isolated in 8/12 , followed by a. flavus in 2/12 and fusarium solani in 1/12 . the presenting visual acuity ranged from light perception ( lp ) to counting fingers . the visual acuity at final follow - up was 20/400 or better in 7/12 eyes ( 58.33% ) and 20/60 in 2/12 eyes ( range 20/60 to lp ) . all eyes with corneal involvement had final visual acuity 20/400 or worse . globe salvage was achieved in all cases.conclusioncombining intravitreal amphotericin - b and voriconazole could be a novel treatment strategy in the management of endophthalmitis caused by filamentous fungus . eyes with corneal involvement had poor visual outcome either with or without therapeutic penetrating keratoplasty .	has been reported in the successful management of refractory systemic aspergillus infections.5 however , there are reports of disparity in results for the in vivo and in vitro effects of combining the two different groups of antifungal drugs.6,7 we report our experience with use of a combination of intravitreal amphotericin b and voriconazole in the management of 12 consecutive cases of filamentous fungal endophthalmitis . this was a retrospective study of 12 consecutive endophthalmitis cases caused by filamentous fungi that were managed at our center between january 2013 and august 2013 . medical records were reviewed in accordance with the guidelines laid down by the declaration of helsinki . the data collected consisted of demographic details , affected eye , etiology , duration of symptoms , clinical characteristics , visual acuity and intraocular pressure at presentation and at subsequent follow - up . the collected samples , which included corneal scrapings , vitreous biopsy , and explanted intraocular lens , were plated in both bacterial and fungal culture media . microbiology records were reviewed for intraocular samples tested , direct microscopy results , and culture characteristics . the details of the treatment course , including time and number of surgical interventions , along with type , number , route , and duration of use of antifungals , were recorded ( table 1 ) . twelve consecutive cases of filamentous fungal endophthalmitis were included in this single - center case series . details of demographic features , clinical characteristics , microbiology results , treatment , and clinical outcome for all cases are described in table 1 . the patients presented to us at a median 22 ( mean 28.4 , range 2262 ) days postoperatively . nine post cataract surgery endophthalmitis cases reported onset of symptoms within one week of surgery . however , patients originating from a remote rural area presented with a delay of 2162 days after surgery . two endophthalmitis cases occurred following open globe injury and one following keratitis after pterygium excision . the presenting complaint was a sudden - onset decrease in visual acuity in eleven eyes ( 91.6% ) associated with pain in eight eyes ( 66.6% ) . four eyes had associated corneal or scleral tunnel infiltrate , and fungal hyphae were demonstrated on microscopy for all of these . six eyes ( 50% ) presented with hypopyon or anterior chamber exudates , while all eyes had exudates in the vitreous cavity that were either seen clinically or demonstrated on b - scan ultrasonography . all eyes underwent pars plana vitrectomy with injection of antifungals , comprising daily intravitreal voriconazole ( 100 g/0.1 ml ) and alternate day intravitreal amphotericin b ( 5 g/0.1 ml ) , known as the amb - vo regimen , until resolution of vitreous exudates . the decision to inject antifungals was based on detection of fungal hyphae on smears from corneal scrapings or clinical suspicion of fungal etiology . all post cataract surgery endophthalmitis cases underwent iol explantation , seven eyes underwent repeat vitrectomy , and three eyes underwent penetrating keratoplasty . systemic antifungal ketoconazole 200 mg twice daily was administered in all cases with corneoscleral involvement . the mean duration of treatment with the amb - vo regimen was 21.5 ( range 1330 ) days . patients with corneal or scleral involvement also received topical 5% natamycin and 1% voriconazole eye drops . globe salvage was achieved in all cases , with a final visual acuity of light perception or better . visual acuity at last follow - up ranged from 20/60 to light perception , with 20/400 or better in seven of the 12 eyes ( 58.3% ) and 20/60 in 2/12 ( 16.6% ) eyes . all eyes had resolution of vitreous exudates as assessed by indirect ophthalmoscopy or by b - scan ultrasonography where the cornea was hazy . the mean presenting and final intraocular pressures were 9.5 ( range 217 ) mmhg and 6.8 ( range 310 ) mmhg , respectively . clinical pictures of the anterior segments from a few patients at presentation and the final visit are shown in figures 1 and 2 . culture - proven fungal endophthalmitis was seen in 11/12 eyes ( 91.6% ) , while one post cataract surgery endophthalmitis case did not grow any organisms ( table 1 , case 3 ) . fungal filaments were seen on direct examination of the vitreous sample ( calcofluor white and gram stain ) in 4/12 ( 33.3% ) cases and in corneal scrapings in 4/12 ( 33.3% ) . significant growth of filamentous fungi was seen in cultures for vitreous samples in 7/12 eyes ( 58.3% ) and explanted intraocular lens plated on chocolate agar in 7/12 eyes ( 58.3% , table 1 ) . characteristic fungal colonies , either cinnamon brown , powdery , or yellowish green , granular were grown , which were suggestive of aspergillus terreus and aspergillus flavus , respectively . the vitreous from the single case of fusarium endophthalmitis showed a creamy , fluffy pinkish colony and spores characteristic of fusarium on microscopy . fungal endophthalmitis is often refractory to antifungal therapy , with poor functional and anatomic outcomes.5,6 the principal objective of this study was to evaluate the role of combined intravitreal amphotericin b and voriconazole in the management of endophthalmitis caused by filamentous fungi . in the current study , we treated filamentous fungal endophthalmitis with pars plana vitrectomy and a combination of intravitreal antifungal agents . this antifungal combination regimen was based on our clinical experience with case 1 ( table 1 ) . this patient had undergone an open globe injury repair followed by pars plana lensectomy and vitrectomy for traumatic cataract and vitreous hemorrhage . two months later , a condensed round mass of anterior vitreous exudates was seen clinically on slit - lamp examination through the pupil behind the iris in the inferotemporal quadrant . fungal hyphae were demonstrated in the vitreous aspirate on microscopy and were later identified as a. flavus from growth on culture . based on initial resolution but re - emergence of exudates during this course , we clinically suspected the isolate to be refractory to voriconazole alone and combined it subsequently with intravitreal amphotericin b given every 48 hours . rapid and complete resolution of vitreous exudates was observed at the end of one week ( figure 3 ) . we formulated a regimen comprising a combination of intravitreal voriconazole daily and amphotericin b every 48 hours ( the amb - vo regimen ) . the frequency of injections was based on the half - life of amphotericin b and voriconazole in aphakic vitrectomized eyes being 1.8 days and 2.5 hours , respectively.8,9 the in vivo efficacy and the role of a combination of antifungal agents in life - threatening systemic fungal infections has been established.5 combination of intravitreal antifungals in the management of fungal endophthalmitis with a good visual outcome has been reported previously.10 in vitro studies predicting the interaction of antifungal agents and the result of combining drugs have demonstrated variable patterns of drug interactions , like synergism , indifference , and often antagonism between these agents.6 amphotericin b , a polyene , is the most commonly used empirical antifungal agent in systemic mycosis and endophthalmitis caused by filamentous fungi.11 it acts by binding to surface sterols in the cell membrane of the fungi , resulting in formation of pores and altered permeability.8 voriconazole , an azole , acts chiefly by depleting ergosterol , the chief bioregulator of membrane integrity.11 steinbach et al hypothesized that azoles may render amphotericin b inactive or be adsorbed onto the fungal cell surface and inhibit binding of amphotericin b to the fungal cell membrane.7 amphotericin b , when combined with lipophilic triazoles like itraconazole , was found to be deleterious or ineffective , while amphotericin b when combined with voriconazole was found to be beneficial in a few but not all experimental studies.57 although filamentous fungus developing resistance to amphotericin b or having inherent resistance has been reported , the frequency has not been described.11,12 a. terreus isolated from the endophthalmitis outbreak in the current series has also been reported to be refractory to amphotericin b in in vitro studies.12 wykoff et al have described fungal isolates , treatment strategies , and clinical outcomes in cases with exogenous fungal endophthalmitis.13 systemic antifungals were required in 83% of patients , with additional intraocular antifungals needed in 95% of eyes . fifty - nine percent of patients were treated with multiple antifungal agents and 20% received anti - fungal agents for 36 months . fifty - four percent of patients achieved a final visual acuity of 20/400 or better while 24% eyes lost perception of light including 20% that underwent enucleation . amongst the various categories , 54% of patients with postoperative fungal endophthalmitis achieved a visual acuity of 20/80 or better , while none of the cases with fungal endophthalmitis following open globe injury achieved vision better than 20/400 , and 70% of these underwent enucleation . narang et al reported poor visual and anatomic outcomes in eyes with exogenous fungal endophthalmitis , especially when associated with corneal involvement.2 in the current study , at a mean final follow - up of 12.75 weeks , the best corrected visual acuity achieved was 20/400 or better in 7/12 eyes ( 58.33% ) and 20/60 in 2/12 eyes ( 16.6% ) . all of the five eyes with corneal involvement developed hypotony and corneal haze or graft opacification with visual acuity of 20/400 or worse . five of six eyes with persistent hypotony had final visual acuity less than 20/400 at last follow - up , with only one such eye recovering a visual acuity of 20/60 . unlike previous reports of poor visual and anatomic outcomes in fungal endophthalmitis following open globe injury,13 patients in the current study recovered a visual acuity of 20/100 or better at the last visit . despite a delayed presentation ( mean 27.6 days ) , 5/9 ( 55.5% ) patients who were part of a postoperative outbreak of endophthalmitis caused by a. terreus finally recovered visual acuity of 20/400 or better . the main limitations of this study are its retrospective design and limited follow - up period . further , all patients could not afford systemic antifungal drugs . due to the paucity of such cases , a prospective randomized controlled trial would be difficult to perform , but a head - to - head comparison between voriconazole alone and a combination of voriconazole and amphotericin b would be useful for establishing which regimen is better . the majority ( 75% ) of isolates in the current series were a. terreus , which has previously been reported to be inherently resistant to amphotericin b.12 it would be useful to study the effect of combining amphotericin b and voriconazole in vitro with regard to possible synergism and correlate it with the in vivo response . in conclusion , combination intravitreal amphotericin b and voriconazole injections until complete resolution of vitreous exudates could be a promising modality of treatment in the management of exogenous filamentous fungus endophthalmitis .
watershed infarcts ( wi ) evolve in hemodynamic risk zones . clinical picture of wi can be associated to partial epileptic seizures . diffusion weighted brain magnetic resonance imaging ( mri ) allows a clear diagnosis . wi pathogenesis involves either embolic or hemodynamic mechanism . a 69-year old patient presented with sub - acute occurrence of right hemiparesis and partial epileptic seizures of the right arm . carotid ultrasounds demonstrated occlusion of the right extra - cranial internal carotid artery ( ica ) and tight stenosis of the contralateral ica . brain diffusion - weighted magnetic resonance revealed acute ischemic lesions within the watershed area of the left hemisphere . our case supports the hypothesis of impaired washout of emboli in low - perfusion brain areas as the mechanism underlying cortical wi .	cerebral watershed ( or border zone ) infarcts ( wi ) involve the junction of the distal fields of two non - anastomosing arterial systems , which are hemodynamic risk zones . there are two types of wi : cortical wi , occurring at the junction between cortical territories of the anterior , middle and posterior cerebral arteries ; and internal wi , occurring in the white matter between the deep and superficial perfusion systems of the middle cerebral artery . clinical picture of watershed infarctions is often progressive or fluctuating ; symptoms may be mild and are therefore probably underestimated . another characteristic feature is occurrence of early - onset partial seizures , which in cortical wi occur more often than in other forms of stroke . due to its sub - acute onset and to the association to partial clonic seizures , wi diagnosis can be challenging for physicians . nevertheless , wis account for up to 10% of all cerebral infarcts and they must be kept under consideration in order to avoid inappropriate treatment in the acute phase . in this phase , diffusion weighted brain mri ( dwi ) allows a clearer diagnosis than cranial computed tomography ( ct ) . we report on the case of a 69-year - old man who presented to the emergency department of our hospital four days after the sub - acute occurrence of homonymous right hemianopia and right hemiparesis . the patient 's medical history disclosed smoking habit ( 78 pack years beginning at an age of 16 years ) . one year earlier , a non - small cell lung carcinoma was diagnosed and treated consequently with 6 cycles of palliative chemotherapy ( carboplatin and gemcitabin ) . neurological examination on hospital admission revealed paresis of the right upper limb and a severe motor deficit of the right lower limb with intact sensibility . the patient was admitted with a diagnosis of ischemic stroke . and was started on secondary prevention therapy according to international guidelines for ischemic stroke treatment with acetylsalicylic acid 300 mg daily . a few hours after hospital admission , the patient developed partial epileptic seizures of the right upper limb , which required iv phenytoin infusion . carotid ultarsounds demonstrated occlusion of the right extra - cranial carotid artery and tight stenosis of the contralateral internal carotid artery ( figure 1a , b ) . brain diffusion - weighted magnetic resonance ( dwi ) revealed multiple hypertintense lesions extending within the cortical watershed area of the left hemisphere , suggesting a large watershed infarct ( figure 1c , d , e ) . figure 1duplex scan showing ( a ) tight stenosis of the left extra - cranial internal carotid artery and ( b ) occlusion of the right internal carotid artery . diffusion weighted brain - magnetic resonance imaging ( mri ) showing watershed infarcts within the left hemisphere ( c , d ) ; mr angiography showing hypo - perfusion of the right intra - cranial internal carotid artery ( e ) . duplex scan showing ( a ) tight stenosis of the left extra - cranial internal carotid artery and ( b ) occlusion of the right internal carotid artery . diffusion weighted brain - magnetic resonance imaging ( mri ) showing watershed infarcts within the left hemisphere ( c , d ) ; mr angiography showing hypo - perfusion of the right intra - cranial internal carotid artery ( e ) . over the following days , the patient had a nearly full clinical recovery , with a residual slight motor deficit of the distal right upper limb at discharge . surgical or percutaneous treatment of left carotid stenosis was excluded in consideration of his advanced neoplastic disease ; he continued adequate medical treatment with antiplatelet therapy acetylsalicylic acid 300 mg daily and statin . we report on a typical neurological picture of cortical wi , which represents a complex clinical entity . our patient sought for medical help only four days after symptoms onset , due to slow progression of the neurological deficit . this was associated to partial epileptic seizures of the upper limb , which are common in cortical wi and display a good response to antiepileptic treatment . another typical feature displayed by the present case was the benign clinical course of the ischemic event , which is often seen in cortical wi . sub - acute onset and association to partial seizures could make the diagnosis of stroke quite challenging . dwi proved to be the most useful imaging technique in order to make a right diagnosis in the acute phase of the disease and to understand its pathogenesis . based on the fact that wi may occur during marked systemic hypoperfusion , the supposed pathological mechanism is hemodynamic failure . this theory is supported by the typical localization of wi in low - perfusion cerebral areas , also called distal or border zone fields . moreover , syncope at wi onset , which is occasionally reported , might support the hemodynamic hypothesis . as opposed to the hemodynamic hypothesis , either cardiac or artery - to artery or microembolism various neuropathological and imaging studies found multiple embolic occlusions in the terminal vascular field distal to atherosclerotic carotid plaques , in particular in the cortical form of wi . anyway , a combination of both theories is likely to be the most realistic mechanism , in terms of impaired washout of emboli in case of decreased perfusion . our patient presented with a cortical wi distal to a tight stenosis of the left internal carotid artery associated to occlusion of the right one . hence , hypoperfusion could definitely play a substantial role in the genesis of the reported case . nevertheless , we suggest that this case could support the hypothesis of an impaired washout of emboli in low - perfusion brain areas . indeed , if hypoperfusion was to be the dominant mechanism of the ischemic lesion , watershed area distal to the occluded carotid artery should to have been at even higher risk . on the other hand , our case keeps in line with the concept that embolic more than hemodynamic mechanism plays a crucial role in the pathogenesis of cortical wi . carotid reperfusion would have been an option in this case , although medical therapy alone was the preferred choice due to the high risk of the procedure in this patient and his global prognosis , due to his underlying disease . wi are a frequent clinical entity , accounting for up to 10% of all cerebral infarcts , although they are often under - recognised due to slow progression of the presenting symptoms and to the frequent association to seizures . our case is a very illustrative example of cortical wi with typical onset , course and imaging appearance . we suggest that it supports the hypothesis of impaired washout of emboli in low - perfusion brain areas in cortical wi ; treatment should be chosen accordingly .
[ purpose ] to investigate the effect of tai chi on cognitive and physical function in the elderly . [ subjects and methods ] a randomized trial design was used . a total 150 subjects were enrolled and were divided into tai chi and control groups . subjects in the tai chi group participated tai chi for 6 months , and subjects in the control group participated in other non - athletic activities . [ results ] there were no differences between the groups in the one leg standing time with eyes open , left grip strength , or the frontal assessment battery at bedside after 3 and 6 months of intervention . the mini - mental state examination scores after 3 and 6 months were higher in the tai chi group than in the control group . the right grip strength after 3 months increased more in the tai chi group than in the control group . both the 5-m high walking speed and 10-m normal walking speed were significantly lower after 3 and 6 months of tai chi practice . [ conclusion ] these results suggest that regular tai chi practice may improve cognitive and physical function in the elderly .	dementia is closely associated with aging , and cognitive decline is common in the elderly . in 2010 , the number of people diagnosed with dementia was 35 million , and the population is projected to increase to 115 million by 20501 . at present , 8.87% of people in china are older than 65 years of age , and the population is expected to rapidly increase to 22.6% by 2050 . in china , 12% of dementia patients are 6069 years old , 48% are 7079 years old , and 1520% are more than 85 years old . the population of elderly people with dementia in china is expected to total 20 million by 20252 . in recent years , interventions to reduce cognitive dysfunction have been investigated , including memory therapy3 , music therapy4 , and exercise therapy5 . in china , therapeutic exercise has been used as a community nursing intervention , but it is limited to simple exercises such as walking6 . tai chi is a traditional chinese martial art and is widely practiced by chinese people . many studies have shown that tai chi has beneficial effects on balance , falls , and non - vertebral fractures7,8,9 . tai chi also reportedly improves renal function in patients with chronic renal disease and cardiac function in cardiovascular disease patients via regulation of lipid metabolism10 . many studies have investigated methods to prevent dementia symptoms , but few have investigated the effect of tai chi on dementia11 , 12 . this study aims to investigate whether tai chi can improve physical and cognitive function in elderly chinese subjects . a total 150 fifty elderly people residing in the local communities were recruited by public announcement . the inclusion criteria were as follows : ( 1 ) > 60 years of age ; ( 2 ) mini - mental state examination ( mmse ) score > 24 , indicating no cognitive impairment ; ( 3 ) sufficient upper limb mobility to perform requisite finger - pointing tasks , such as flexing and extending the shoulder , elbow , wrist , and fingers ; and ( 4 ) no neurologic or musculoskeletal disease such as stroke or visual and auditory impairments . subjects meeting the inclusion criteria were randomly allocated to the tai chi group or the control group using a random number table ( n = 75 per group , fig . 1fig . 1.study protocol and subject recruitment . a total 150 subjects > 60 years old were randomly divided into a tai chi intervention group and a control group . all participants were investigated at baseline and at 3 and 6 months after beginning intervention . ) . an independent researcher blinded to the number allocation enrolled and assigned subjects to each group . study protocol and subject recruitment . a total 150 subjects > 60 years old were randomly divided into a tai chi intervention group and a control group . all participants were investigated at baseline and at 3 and 6 months after beginning intervention . subjects in the tai chi group practiced 24-form yang style tai chi for 60 minutes twice weekly for 6 months . the control group participated in other activities such as playing cards or singing at the activity center . however , due to a variety of factors such as hospitalization , the intervention group included 72 people after 3 months and the control group 66 people after 6 months . after completing the study , the control group was provided a similar intervention program . before the study , all subjects were given 10 min to read a document on dementia prevention , and a researcher answered any questions concerning the document . each subject completed a questionnaire concerning gender , age , education , disease status , and the functional capacity tokyo metropolitan institute of gerontology index of competence ( tmig , 013 points ) . the tmig is used to measure the functional capacity in daily life ; higher scores indicate greater functional ability13 . physical function was assessed using the eyes open / leg standing time , 5-m high walk speed , 10-m normal walk speed , left grip strength , and right grip strength14 . the subjects were asked to stand on either leg as long as possible while keeping their eyes open to assess their static balance . the time from when the subject raised a leg until the raised leg touched the ground was measured twice , and the longer time of the two trials was analyzed . grip strength was measured twice in both hands using an adjustable handheld dynamometer to assess muscular strength , and the highest measure for each hand was used . participants were asked to walk 10 m at normal speed or 5 m at maximal speed , and the mean time was measured using a stopwatch . cognitive function was measured using the mmse ( 030 points ) and frontal assessment battery ( fab ) at bedside ( 018 points)15 , 16 . for the mmse , 2324 points indicates mild dementia , while higher scores indicate better cognitive function . for the fab at bedside , data were analyzed using the test before and after intervention in the tai chi group and control group . statistical analysis was performed using spss 13.0 , and p < 0.05 was considered statistically significant . of these , 138 subjects completed the 6-month study program , and 12 subjects ( tai chi group n = 3 , control group n = 9 ) were lost to follow - up . as a result , follow - up data were available for 72 of 75 subjects in the tai chi group and 66 of 75 subjects in the control group . table 1table 1.subjects characteristicsparametertai chi group , n = 72 ( % ) control group , n = 66 ( % ) gendermale14 ( 19.4)20 ( 30.3)female58 ( 80.6)46 ( 69.7)age ( years)mean sd68.3 5.970.1 5.7606422 ( 30.6)12 ( 18.2)656922 ( 30.6)20 ( 30.3)707416 ( 22.2)18 ( 27.3)757910 ( 13.9)14 ( 21.2)80842 ( 2.8)2 ( 3.0)education ( years)01132 ( 44.5)18 ( 27.2)1240 ( 55.5)48 ( 72.8)diseasecardiac16 ( 22.2)8 ( 12.1)hyperpiesia12 ( 16.7)16 ( 24.2)diabetes mellitus4 ( 5.6)4 ( 6.1)other18 ( 25.0)22 ( 33.3)none22 ( 30.6)16 ( 24.2)physical exerciseyes54 ( 75.0)54 ( 81.8)no18 ( 25.0)12 ( 18.2)exercise frequency<3/w22 ( 40.7)20 ( 47.0)3/w32 ( 59.3)34 ( 63.0)exercise time ( min)<6038 ( 70.4)36 ( 66.6)6016 ( 29.6)18 ( 33.4)tmig ( 413 points)mean sd11.6 1.310.8 2.1physical functionone leg standing time with eyes open ( s)28.7 27.123.3 23.05-m high walking speed ( s)2.9 0.72.8 0.810-m normal walking speed ( s)8.7 2.18.9 2.5left grip strength ( kgw)24.7 6.923.6 10.3right grip strength ( kgw)25.5 7.023.9 10.5brain functionmmse ( 2030)26.4 2.426.8 1.7fab ( 718)14.6 2.114.1 2.9tmig ; functional capacity tokyo metropolitan institute of gerontology index of competence : mmse ; mini - mental state examination : fab ; frontal assessment battery at bedside : sd ; standard deviation , significant difference between groups ; the two groups were well matched at the baseline assessment , and there were no obvious differences in the key outcome variables initially , except the tmig score , which was significantly higher in the tai chi group than in the control group ( p < 0.01 ) . tmig ; functional capacity tokyo metropolitan institute of gerontology index of competence : mmse ; mini - mental state examination : fab ; frontal assessment battery at bedside : sd ; standard deviation , significant difference between groups ; p < 0.01 in the tai chi group , the tmig score increased at both 3 and 6 months . the eyes opened / one leg standing time and fab increased at 6 months , but were unchanged at 3 months . by contrast , the right grip strength increased at 3 months , but not at 6 months . there were no significant differences in the eyes open / one leg standing time , left grip strength , and fab at bedside ( p > 0.05 ) between the tai chi and control groups at 3 or 6 months ( tables 2table 2.physical and cognitive functions compared between the tai chi and control groupsparametertai chi ( n = 72 ) mean ( sd)control ( n=66 ) mean ( sd)pre - intervention3 months6 monthspre - intervention3 months6 monthstmig ( 413)11.6 ( 1.3)12.1 ( 0.9)12.1 ( 0.9)10.8 ( 2.1)11.1 ( 2.0)11.7 ( 1.4)physical exerciseone leg standing time with eyes open ( s)28.7 ( 27.1)36.9 ( 38.3)40.0 ( 47.1)23.3 ( 23.0)34.3 ( 37.5)38.0 ( 40.7)5-m high walking speed ( s)2.9 ( 0.7)2.8 ( 0.5)2.8 ( 0.7)2.8 ( 0.8)3.2 ( 0.8)3.4 ( 0.9)10-m normal walking speed ( s)8.7 ( 2.1)8.5 ( 2.0)8.6 ( 1.6)8.9 ( 2.5)9.7 ( 2.6)9.6 ( 2.6)left grip strength ( kgw)24.7 ( 6.9)24.9 ( 6.5)24.9 ( 8.8)23.6 ( 10.3)23.1 ( 11.5)22.8 ( 10.3)right grip strength ( kgw)25.5 ( 7.0)26.7 ( 7.2)25.8 ( 7.7)23.9 ( 10.5)23.4 ( 11.5)23.4 ( 11.0)brain functionmmse ( 2030)26.4 ( 2.4)27.8 ( 1.7)28.0 ( 2.0)26.8 ( 1.7)27.1 ( 1.8)27.3 ( 1.9)fab ( 718)14.6 ( 2.1)14.9 ( 2.4)15.7 ( 2.4)14.1 ( 2.9)14.3 ( 2.8)14.5 ( 3.2)tmig ; functional capacity tokyo metropolitan institute of gerontology index of competence : mmse ; mini - mental state examination : fab ; frontal assessment battery at bedside : sd ; standard deviation , * significant difference between groups ; p < 0.001 , p < 0.01 , p < 0.05 , 3table 3.outcome variables after 3 and 6 months tai chi training3 months after intervention6 months after interventiontai chi group(n = 72)mean ( sd)control group(n = 66)mean ( sd)tai chi group(n = 72)mean ( sd)control group(n = 66)mean ( sd)tmig ( 413)0.5 ( 1.0)0.5 ( 1.5)0.5 ( 1.2)0.9 ( 1.3)physical exerciseone leg standing time with eyes open ( s)8.1 ( 35.5)11.0 ( 26.1)11.3 ( 45.0)14.8 ( 28.9)5-m high walking speed ( s)0.1 ( 0.5)0.4 ( 0.5)0.1 ( 0.7)0.6 ( 0.7)10-m normal walking speed ( s)0.2 ( 2.2)0.8 ( 1.3)0.1 ( 1.6)0.8 ( 1.7)left grip strength ( kgw)0.2 ( 2.9)0.5 ( 3.5)0.2 ( 4.3)0.7 ( 3.3)right grip strength ( kgw)1.2 ( 3.2)0.5 ( 4.1)0.2 ( 3.1)0.5 ( 3.5)brain functionmmse ( 2030)1.4 ( 2.2)0.3 ( 2.2)1.6 ( 1.9)0.5 ( 2.6)fab ( 718)0.3 ( 1.8)0.3 ( 2.6)1.1 ( 2.2)0.5 ( 2.6)tmig ; functional capacity tokyo metropolitan institute of gerontology index of competence : mmse ; mini - mental state examination : fab ; frontal assessment battery at bedside : sd ; standard deviation , significant difference between groups ; p < 0.001 , p < 0.01 , p < 0.05 ) . tmig ; functional capacity tokyo metropolitan institute of gerontology index of competence : mmse ; mini - mental state examination : fab ; frontal assessment battery at bedside : sd ; standard deviation , * significant difference between groups ; p < 0.001 , p < 0.01 , p < 0.05 tmig ; functional capacity tokyo metropolitan institute of gerontology index of competence : mmse ; mini - mental state examination : fab ; frontal assessment battery at bedside : sd ; standard deviation , significant difference between groups ; p < 0.001 , p < 0.01 , p < 0.05 participants in the tai chi group had a significantly decreased 5-m high walking speed ( p < 0.001 ) , 10-m normal walking speed ( p < 0.01 ) , right grip strength ( p < 0.01 ) , and mmse ( p < 0.01 ) compared with the control group at 3 months . furthermore , the tai chi group experienced a significantly decreased 5-m high walking speed ( p < 0.001 ) , 10-m normal walking speed ( p < 0.001 ) , and mmse ( p < 0.01 ) compared with the control group at 6 months ( table 2 ) . the 5-m high walking speed test decreased in the tai chi group at both 3 and 6 months , while the control group showed an increasing trend ( tables 2 , 3 ) . a similar trend was observed for the 10-m normal walking speed at 3 and 6 months ( tables 2 , 3 ) . the right grip strength significantly increased after 3 months but decreased at 6 months in the tai chi group . in the control group , the right grip strength decreased at both 3 and 6 months ( tables 2 , 3 ) . finally , the mmse score increased at both 3 and 6 months in the tai chi group ( tables 2 , 3 ) . this study shows that tai chi practice improves cognitive and physical functions in the elderly . we found that the mmse score significantly increased in the tai chi group compared with the control group , suggesting that tai chi may improve the mental function of the elderly in china . exercise is known to increase regional cerebral perfusion in the contralateral motor sensory cortex , which may explain why tai chi may increase memory and concentration17 . although an increasing number of elderly chinese people practice tai chi , few studies have examined the effects of tai chi on preventing dementia . brown et al.18 randomly divided 135 subjects into a tai chi group and control group for 16 weeks of moderate and low intensity exercise programs , and found that exercise with tai chi was more effective than exercise alone in promoting psychological benefits in both men and women . winter and tse19 , 20 showed that tai chi could improve walking speed and grip strength in the elderly . consistent with these previous reports , in this study , we found that walking speed was significantly improved in the tai chi group compared with the control group . in addition , schaller21 reported that tai chi practitioners had significantly better eyes open / leg standing times . however , in this study we found that the eyes open / one leg standing time significantly increased in both the tai chi and control groups after 3 and 6 months , and there was no difference between the two groups . of note , the participants in the control group in schaller s study maintained their current activity level , while the control group in this study participated in non - athletic activities such as playing cards or singing . our results also showed a significantly increased tmig in both the tai chi and control groups . however , there was no difference between the two groups , which may be because all of the subjects read a document about dementia prevention before beginning the study . currently , family care and community support systems are not well established in china . therefore , it is particularly important to take precautions for the prevention and early treatment of dementia . presently , there are few intervention studies in china on dementia prevention by tai chi . we conducted a randomized controlled trial to evaluate the potential to prevent dementia using tai chi , and our results demonstrate that tai chi can improve cognitive function in elderly people . many studies , especially those evaluating older adults or significantly impaired individuals , have reported functional changes over 3 to 6 months following intervention , but the six - month duration of this study only evaluated the potential effect of tai chi on age - related dementia . tai chi is traditionally considered a life - long skill , and it is believed that with time , both proficiency and health benefits improve . long - term interventional studies and well - designed observational studies comparing long - term tai chi practitioners ( e.g. > 10 years ) to matched controls are needed to fully evaluate the long - term impact of tai chi on healthy aging22 . in conclusion , our results confirmed that tai chi is potentially useful in preventing dementia in elderly people . tai chi could be an easy and effective preclinical treatment strategy of preventing dementia in elderly people and enhancing the quality of life .
to detect orthopoxvirus in the brazilian amazon , we conducted a serosurvey of 344 wild animals . neutralizing antibodies against orthopoxvirus were detected by plaque - reduction neutralizing tests in 84 serum samples . amplicons from 6 monkey samples were sequenced . these amplicons identified vaccinia virus genetically similar to strains from bovine vaccinia outbreaks in brazil .	during february 2001september 2002 , we captured 344 wild mammals in an overflow area in a fauna - rescue program during construction of a hydroelectric plant in lajeado and ipueiras counties ( 94458s , 482123w ) in tocantins state , brazil . during this program , 269 capuchin monkeys ( cebus apella ) , 27 black - howling monkeys ( allouata caraya ) , 12 coatis ( nasua nasua ) , 20 agoutis ( dasyprocta sp . ) , 2 opossums ( didelphis albiventris ) , 5 armadillos ( euphractus sexcinctus ) , 5 collared anteaters ( tamandua tetradactila ) , and 4 gray foxes ( cerdocyon thous ) were captured . all animals were captured in a sylvatic area and did not have contact with humans and dairy cattle . in field - screening laboratories , animals were then released in areas selected during environmental conservation programs . until 2002 , bovine vaccinia had been restricted to southeastern brazil , > 1,400 km from the study area ( 7 ) . serum samples were inactivated by heating at 56c for 30 min , and an orthopoxvirus plaque - reduction neutralizing test ( prnt ) was performed . prnt was used rather than elisa because secondary antibodies required for an elisa for all analyzed species were unavailable . inactivated samples were diluted 1:201:1,640 in minimal essential medium and tested in vero cells by using the vacv - western reserve strain in the prnt as described ( 8) . human samples positive for antibodies to orthopoxvirus obtained during bovine vaccinia outbreaks were used as positive controls ( 9 ) ; samples negative for these antibodies were used as negative controls ( 10 ) . serum titer was defined as the highest dilution that inhibited > 50% of viral plaques compared with negative controls . orthopoxvirus prnt specificity ( 97.4% ) and sensitivity ( 93.5% ) were confirmed by using receiver - operating characteristic analysis , which compared results of prnt , elisa , and clinical symptoms during bovine vaccinia outbreaks ( 9,10 ) . of 269 c. apella samples , 68 ( 25.3% ) had antibodies to orthopoxvirus . of 27 a. caraya samples , 13 ( 48.1% ) had antibodies to orthopoxvirus . seropositivity was detected in 2 ( 16.6% ) coatis and 1 ( 5.0% ) agouti . of 344 animals studied , 84 ( 24.4% ) had antibodies to orthopoxvirus ( table ) . in samples with high neutralizing antibody titers , 55.95% ( 47 ) had titers of 80320 . only 5 ( 6.0% ) prnt - positive samples had titers < 40 ( table ) . given the serologic cross - reactivity of orthopoxvirus ( 3 ) , positive samples could indicate any of > 9 virus species , although it is well established that vacv is endemic to brazil , and infections with other orthopoxviruses are geographically restricted to other continents and have not been identified in brazil . therefore , we performed a molecular investigation to identify orthopoxviruses associated with orthopoxvirus sylvatic circulation . serologic and molecular tests were performed in a blinded fashion and in triplicate . on the basis of previous studies that detected orthopoxvirus dna in serum of infected hosts ( 9,11,12 ) , a semi - nested pcr was used to amplify the highly conserved orthopoxvirus vaccinia growth factor ( vgf ) gene ( j.s . human vacv dna positive and dna negative serum samples obtained during bovine vaccinia outbreaks ( 9 ) were used as positive and negative controls , respectively . eighteen of 344 serum samples were positive in pcr assays ( 11 from c. apella and 7 from a. caraya ; all were prnt positive ) . six of the 18 vgf pcr - positive samples were chosen for sequencing and analysis of vgf ( 4 from c. apella and 2 from a. caraya ) . ( 14 ) , we amplified the hemagglutinin ( ha ) gene from 2 samples ( 1 from c. apella and 1 from a. caraya ; both were vgf positive ) . the vgf and ha pcr products were cloned into the pgemt - easy vector ( promega , madison , wi , usa ) . three clones from distinct pcr amplicons of each sample were sequenced in both orientations by using m13 universal primers and the mega - bace - sequencer ( ge healthcare , little chalfont , uk ) . optimal alignment of the highly conserved vgf gene with clustalw ( www.ncbi.nlm.nih.gov/pmc/articles/pmc308517 ) and mega version 3.1 ( www.megasoftware.net ) showed 100% identity among all nucleotide and amino acid sequences for monkey serum ( appendix figure , panel a ) . when compared with nucleotide sequences available in genbank , vgf sequences were highly similar ( 98%100% identity ) to the homologous gene from other vacv strains and showed 100% identity . the ha sequences for c. apella and a. caraya showed a signature deletion ( appendix figure , panel b ) also present in sequences of other vacv isolates from brazil . these ha sequences showed 99.6% identity at the nucleotide level and 99.7% identity at the amino acid level ( 736 nt of the ha gene were analyzed ) . phylogenetic trees of the vgf ( figure , panel a ) or ha ( figure , panel b ) genes were constructed by using the neighbor - joining method , 1,000 bootstrap replicates , and the tamura 3-parameter model ( mega version 3.1 ) . the vgf and ha sequences from monkey samples were deposited in genbank ( accession nos . consensus bootstrap phylogenetic trees based on nucleotide sequences of orthopoxvirus vaccinia growth factor ( vgf ) ( a ) and hemagglutinin ( ha ) ( b ) genes . trees were constructed with ha or vgf sequences by using the neighbor - joining method with 1,000 bootstrap replicates and the tamura 3-parameter model in mega version 3.1 software ( www.megasoftware.net ) . black dots indicate vaccinia virus ( vacv ) obtained from cebus apella ( vacv - to ca ) and allouata caraya ( vacv ac ) . all vgf sequences obtained from monkey serum samples showed 100% and are represented as a unique sequence in the vgf tree ( vacv to ) . hspv , horsepoxvirus ; varv , variola virus ; cpxv , cowpoxvirus ; mpxv , monkeypoxvirus . although vacv strains have been isolated from rodents in forests in brazil ( 5,6 ) ( the nearest location , belm , is 750 km from the study area ) , we detected vacv in wildlife in the brazilian amazon 3 years after reports of exanthematic outbreaks of bovine vaccinia and 40 years after isolation of vacv from forests . our data provide evidence of high prevalence of orthopoxviruses among capuchin and black - howling monkeys in the brazilian amazon . the relationship between infected monkeys and emergence of vacv in rural regions of brazil is unknown . however , transmission of vacv in northeastern brazil has been reported , and outbreaks have been reported in mato grosso , pernambuco ( www.amep.org.br/pox.doc ) , maranho ( e.g. kroon et al . , unpub . data ) , and tocantins ( 13 ) , which are in or adjacent to the brazilian amazon . some of these viruses may be related to those isolated in this study because some vacv isolates have the same signature deletion in the ha gene as vacv - to . anthropogenic disturbance of the amazon ecosystem and increases in agricultural and livestock areas increase contact between wildlife and rural populations ( 15 ) . however , the effect of vacv in environments in brazil that contain wild animals has not been studied . clinical data for pox lesions in animals tested were not well documented by veterinarians in the study area . ecologic and public health studies should be designed to evaluate risks for infection with vacv during wildlife conservation efforts and determine whether surveillance systems can predict bovine vaccinia outbreaks by monitoring vacv infection in monkeys and other wild animals . amino acid sequences of vaccinia virus ( vacv ) samples and comparison with homologous genes sequences from several orthopoxviruses , brazil . a ) alignment of vaccinia growth factor gene sequences from 6 monkey serum samples showing 100% identity ( horizontal box ) . vacv - to_ca , sequence from cebus apella ; vacv - to_ac , sequence from allouata caraya ; hpxv , horsepoxvirus ; cpxv , cowpoxvirus ; mpxv , monkeypoxvirus ; varv , variola virus ; ecmv , ectromelia virus . b ) alignment of orthopoxvirus hemagglutinin gene amino acid sequences showing the deletion signature region ( vertical box ) in vacv - to isolates and several vacv strains isolated during bovine vaccinia outbreaks . arrow indicates polymorphism site in the hemagglutinin amino acid sequences between vacv - to_ca and vacv - to_ac . alignments were made by using clustalw ( www.ncbi.nlm.nih.gov/pmc/articles/pmc308517 ) and mega version 3.1 software ( www.megasoftware.net ) .
purpose : disabled individuals younger than 65 years are entitled to medicare coverage through the social security disability insurance ( di ) program , but only if they have completed a 2-year waiting period . this is the first study that uses longitudinal panel data , the health and retirement study , and examines whether and to what extent the health and economic status are affected among disability beneficiaries who are uninsured during the medicare waiting period.methods:in a quasiexperiment research design , using a difference - in - difference ( diff - in - diff ) estimator , we compare changes in health and economic outcomes pre-/postentering the di program for disability beneficiaries with alternative public health insurance and those without.results:the adjusted diff - in - diff estimates suggest that disability beneficiaries who are uninsured during the waiting period , compared to those who are insured , are 13.6 percentage point more likely to report poor health , 6.3 percentage point less likely to be in excellent health , declare more difficulties in activities of daily living , and 30% higher medical expenditures from out of pocket.conclusions:the findings highlight punitive health and economic effects of the medicare waiting period for uninsured disability beneficiaries . we also discuss the implications of the findings for the affordable care act reform .	medicare is a federally administered social insurance program in the united states that guarantees access to health insurance for individuals aged 65 and older and younger individuals with disabilities who have worked and contributed to the system . in 2013 , while disability beneficiaries comprise 16.8% of the total medicare population , they account for more than 20% of the total program expenditures . in the last decade , the growth of medicare enrollment from disability beneficiaries has greatly outpaced the overall growth of medicare program . for example , using data from the chronic condition data warehouse medicare 5% sample from centers for medicare and medicaid services , we estimate that from the year 2003 to 2012 , the growth of disabled beneficiaries ( 37% ) is much faster than the growth of the aged beneficiaries ( 21% ) . as baby boomers age , they are reaching an age of increased likelihood to developing disabilities , which makes it more important to understand the disability beneficiaries under medicare . however , younger medicare beneficiaries with work disabilities have received far less research attention and policy consideration than elderly medicare beneficiaries . the disabled individuals receive medicare coverage through the social security disability insurance ( di ) program , a primary public transfer program that provides both partial earnings replacement and medicare coverage to workers who lost earnings capacity due to severe and long - term disabilities . the social security definition of disability refers to severe health limitations that prevent individuals from engaging in any substantial gainful activities and are expected to last at least 12 months . as of december 2013 , the di program provides cash benefits and medicare coverage to nearly 11 million american workers and their eligible dependents . most di beneficiaries are required to complete a 5-month waiting period before they are entitled to cash benefits and an additional 24-month waiting period before they are entitled to medicare coverage ( the 2-year medicare waiting period is waived for those with amyotrophic lateral sclerosis and those with end - stage renal disease ) . there has been considerable concern that the 2-year medicare waiting period causes significant hardship to new di beneficiaries who lack alternative health insurance coverage and needed health care access to address their health problems . evidence shows that more than 20% of di beneficiaries have no health insurance coverage at all during the medicare waiting period . around 1.8 million di beneficiaries congress has noted the problems with the 2-year waiting period in the ticket to work and work incentives improvement act of 1999 . other political effort devoted to changing this policy includes the ending the medicare disability waiting period act of 2007 , sponsored by senator jeff bingaman and rep . gene green , which proposes to phase out medicare s 2-year waiting period over a 10-year span and grant more exceptions to people with life - threatening health conditions . studies based on focus groups and interviews with disabled individuals have shown that many of the uninsured lack needed health care to stabilize their health conditions and their situations get worse during the waiting period . yet formal analyses have been rare that identify and quantify any detrimental effect that the 2-year waiting period has imposed on disabled individuals . a handful of previous studies about the medicare waiting period have primarily focused on estimating the fiscal impact of eliminating the 2-year waiting period , which is certainly useful for budgetary consideration of policy changes . a demonstration project ( the accelerated benefits [ ab ] demonstration ) conducted by the social security administration ( ssa ) in 2011 provides evidence about effects on health care utilization and estimates the fiscal costs once uninsured di beneficiaries are provided with health - care package during the 2-year waiting period . however , the project does not evaluate the impact of health care access on health outcomes for the uninsured disability beneficiaries during the waiting period probably due to the constraint of the project design such as the narrow time window of the follow - up survey ( 6 months ) , which makes it difficult to observe any changes in health outcomes . although the demonstration project provides evidence on some important aspects of providing accelerated health care access to disability beneficiaries , it faces challenges that are endemic to many policy demonstration projects . as the ssa acknowledges , the ab demonstration project is not able to estimate any potential - induced entry effects , which would occur if the accelerated health insurance coverage offered to newly awarded di beneficiaries would make the di program more attractive and induce more applicants to the di program . it is also possible that individuals who expect no health insurance coverage during the medicare waiting period are less likely to apply for di . our study utilizes longitudinal panel data that span over nearly 20 years and allow us to observe disabled workers for a longer period of time and more likely to capture changes in their health and economic status . a lot of research effort has been devoted to examining the impact of medicare on health outcomes . these studies have primarily focused on the medicare impact on elderly beneficiaries rather than disabled beneficiaries . in general , they find that medicare has no discernible effect on health . these studies are helpful for understanding an average effect of medicare on health outcomes among the elderly patients . in the meantime , they may have masked medicare effects on certain subgroups of the population , for example , individuals with poor health . in other words , it is possible that medicare has differential effect on the health distribution . workers younger than 65 who have severe and long - term health impairments and lose earning capacity may qualify for the social security disability benefits through the di program . they are likely more ill and worse off financially on average compared to the elderly beneficiaries who survive age 65 and are entitled to medicare then . although medicare may not show significantly positive health effects for the elderly beneficiaries for their chronic health conditions , as shown in previous literature , it may help improve the health status or at least prevent the health from deteriorating for the disabled beneficiaries for their severe health problems . there is some evidence that for the severely ill patients , acquiring medicare coverage has significant and positive effects on self - reported health , with the largest gains in health improvement among the groups that experience the largest gains in insurance coverage . in this article , we focus on a group of individuals with poor health . they qualify for disability benefits and medicare entitlement through the di program , which adopts the strictest definition of disability compared to the criteria used in most other disability programs in the nation , as well as compared to the disability definition used in disability programs in other developed countries research effort in the past about the di program has mainly focused on understanding the effects of the cash benefits offered by the program . little analysis has been conducted to understanding the effects of the important in - kind benefit , medicare coverage , provided by the di program . the fact that medicare is a federally administered program and its eligibilities are the same for all individuals , limits the extent to which variation in medicare coverage can be used to identify the effects of medicare coverage on individuals in the di program . the medicare waiting period creates a health insurance gap for some disability beneficiaries , while others have alternative health insurance ( mostly medicaid ) in that period . it provides an opportunity for us to exploit variation in health insurance coverage and identify its effects on disabled individuals . this article is the first to take advantage of the longitudinal health and retirement study ( hrs ) and estimate health and economic effects of the medicare waiting period for disabled individuals . a previous study has used the same data source to study the health effects of medicare enrollment at age 65 on the near - elderly uninsured . in this article , we explore whether and to what extent the medicare waiting period impacts health and economic status of the di beneficiaries who are uninsured while waiting for medicare to become available . in a quasiexperiment research design , using a difference - in - difference ( diff - in - diff ) estimator , we compare changes in health and economic outcomes pre-/postentering the di program for the beneficiaries with alternative health insurance and those without during the medicare waiting period . the adjusted diff - in - diff estimates suggest that the disability beneficiaries who are uninsured during the waiting , compared to those who are insured , are 13.6 percentage point more likely to report poor health , 6.3 percentage point less likely to be in excellent health , declare more difficulties in activities of daily living ( adl ) , and about 30% higher medical expenditures from out of pocket . the findings suggest punitive health and economic effects of the medicare waiting period for the uninsured disability beneficiaries . the structure of this article is as follows : the first section introduces the research question . the study uses 10 available waves of the hrs , which cover the 1992 to 2010 period , with some interviews conducted in 2011 . the hrs is a biennial national representative survey and it interviews individuals born between 1931 and 1941 and their spouses , as well as additional cohorts that have been added in recent waves of the study . the data provide extensive information on health status , employment history , wealth , income , family structure , and government program participation and transfers for a total of 30 672 respondents . for our analysis , it is crucial to accurately line up the dates pre-/post - di program for disability beneficiaries and identify the medicare waiting period . one is entitled to di benefits 5 months after he becomes technically disabled according to the social security definition of disability assuming he has become di insured by accumulating enough work credits . he is entitled to medicare coverage 24 months after his entitlement to di benefits . to identify the medicare waiting period , we must determine the date 1 is entitled to di benefits , which could be before the receipt of the first di paycheck . in other words , this is because it can sometimes take many months or even years before a di application is approved . the processing times are even longer for claims that are initially denied and then subsequently allowed during the appeal process . also , those who are eventually allowed benefits might not file a disability claim immediately following the disability onset . hence , by the time di beneficiaries are notified that their claim has been allowed , some have completed all or part of the medicare waiting period . linking the hrs files to the social security master beneficiary record file , we are able to identify the official date of disability onset . the linkage of hrs and master beneficiary files is restricted and available from the university michigan hrs for researchers who meet the criteria for access to confidential data . knowing the date of disability onset , we can count 5 months from that date to determine the date for di entitlement , which is when the medicare waiting period starts . the waiting period ends 29 months after the date of disability onset . in the hrs , that is , a 2-year passing is between the adjacent interview waves in the hrs . comparing the interview dates with the di entitlement date , we designate the wave right prior to the di entitlement the pre - di period and the wave right after the di entitlement the post - di period . we exclude in the sample the respondents for whom we do not observe their pre - di period and those for whom we do not observe the post - di period . so we include in the sample the individuals who are awarded di benefits , for whom we can observe both the wave before and the wave after their di entry ( waves identified based on the interview date and the di entitlement date if available ) , and for whom we can determine whether they have public health insurance coverage in the wave after di entry . after applying all the sample restrictions , we are left with a sample of 465 respondents . in the 2-period model aka equation ( 1 ) , each respondent is observed twice in the wave before entering di and the wave after . in our analysis , we also expand the specification beyond the 2-period pre-/postcomparison to allow multiple pre- and postperiods aka equation ( 2 ) , given the fact that the health effects may not become apparent in a narrow time frame . in the multiperiod model , each respondent contribute up to 4 observations in the waves pre - di entry and up to 4 observations in the waves post - di entry . our goal is to identify the effects of being uninsured during the medicare waiting period for the newly entitled disability beneficiaries . we distinguish the following 2 groups in our analysis : the newly entitled di beneficiaries who do not have any health insurance coverage before medicare becomes available ( 23% of the sample ) and the newly entitled di beneficiaries who have access to alternative public health insurance ( primarily medicaid ) immediately after entering the di program ( 77% of the sample ) . the former group includes the individuals who lose their health insurance after entering the di program ( 14% ) and the individuals who already lose health insurance prior to the di entry ( 9% ) . among the latter , when we drop this latter group from our study sample , the diff - in - diff estimates became slightly larger in magnitude and stayed statistically significant . the di beneficiaries with health insurance in the waiting period include the individuals who gain access to public health insurance once entering the di program ( 57% ) and the individuals who get their public health insurance from before entering the di program ( 19% ) . as we will show later , the 2 groups appear comparable in a large array of socioeconomic characteristics and health variables at the baseline period . the di beneficiaries who have low income and assets may concurrently receive supplemental security income ( ssi ) benefits . most of the di - ssi concurrent beneficiaries gain immediate access to medicaid coverage because of their entitlement to ssi benefits . the 2 public disability transfer programs are administered by the ssa and follow the same criteria for determining disability severity . hence , the beneficiaries under the 2 programs are comparable at the baseline in terms of their disability / health status , a key variable in our analysis . table 1 presents preperiod descriptive statistics for the sample of newly entitled di beneficiaries without health insurance coverage and those with alternative public health insurance upon entering the di program . the disability beneficiaries with alternative public health insurance during medicare waiting period include more women , fewer blacks , and more married . they appear a bit less healthy than those beneficiaries with health insurance in the waiting period . the former report more health conditions and more difficulties in adls and instrumental activities of daily living . the major differences between the 2 groups lie in some of the financial variables . the disability beneficiaries with insurance in the waiting period , compared to those without insurance in the waiting period , on average earns 40% less from work before entering the di program . however , the former receives nonlabor income ( most likely government transfers ) that is 4.5 times the amount received by the latter . these differentials are not surprising as the uninsured beneficiaries consist of disabled individuals who receive di benefits only ( rather than di - ssi concurrently ) and who are most likely workers with longer worker histories and higher labor earnings compared to the insured disabled individuals who are concurrently receiving the ssi benefits that are means tested . sample means / proportions as of the wave prior to di entry for the di beneficiaries with and without health insurance in the waiting period . abbreviations : adl , activities of daily living ; bmi , body mass index ; di , disability insurance ; iadl , instrumental activities of daily living ; prev , previous ; unemp ins and workers comp , unemployment insurance and workers compensation . net worth is the sum of assets ( primary residence , other real estate , vehicles , businesses , iras , stocks , bonds , checking accounts , cds , and other assets ) less liabilities ( mortgages , other home loans , and other debt ) . major health conditions include ( 1 ) high blood pressure or hypertension ; ( 2 ) diabetes or high blood sugar ; ( 3 ) cancer or a malignant tumor or any kind except skin cancer ; ( 4 ) chronic lung disease except asthma such as chronic bronchitis or emphysema ; ( 5 ) heart attack , coronary heart disease , angina , congestive heart failure , or other heart problems ; ( 6 ) stroke or transient ischemic attack ; ( 7 ) emotional , nervous , or psychiatric problems ; and ( 8) arthritis or rheumatism . what is worth noting is that half of the disability beneficiaries have an annual household total income of less than us$30 000 . it suggests that many beneficiaries could benefit from the recently passed health - care reform , the affordable care act ( aca ) , which makes health insurance more affordable by providing subsidies for families with income below 400% of the federal poverty line to purchase insurance through new health insurance exchanges . we will discuss this aspect further in the last section of this article . the underlying theoretical framework for individuals inherit an initial amount of this stock that depreciates with age and can be increased by investment . changes in health stock over time are determined by increased health stock less health depreciation . health stock is produced by health inputs such as medical care , health knowledge , lifestyle , environment , and so on . so in our reduced - form estimation about health changes over time ( pre-/post - di ) , we control for changes in medical care ( health insurance coverage and health - care utilization ) , changes in health knowledge ( proxied by years of schooling , an efficiency parameter in the grossman health production function ) , changes in wealth and income , and other socioeconomic variables as proxy for other factors that may affect health outcomes and hard to measure empirically such as lifestyle . in a quasiexperiment research design , we use a diff - in - diff estimator , where the first difference is between outcome variables before and after entering the di program and the second difference is between the disability beneficiaries with health insurance and those without health insurance in the waiting period . taking advantage of the panel data , we take the within - person change in outcome variables before and after entering the di program . this is desirable as it guarantees comparability of the groups in the pre- and postperiods , controlling for any compositional changes due to attrition or sampling variation . one identifying assumption in diff - in - diff models is that other unobserved time - varying processes would have similarly impacted the 2 comparison groups . the preperiod differences in some measures between the groups in table 1 suggest a potential vulnerability on this front . we address this by testing for differential changes in a number of potential confounders , including health measures , financial variables , and health service utilization . we then present in addition to unadjusted diff - in - diff estimates , the estimates that account for within - person changes in these and other relevant variables . let yijt denote the change in health and economic outcomes for individual i in group j = 1 , 0 ( di beneficiaries with and without insurance in the waiting period ) between time t and t 2 ( 2 years between waves in hrs ) . dj is an indicator for being a di beneficiary with insurance in the waiting period . we begin with a 2-period model in which t 2 denotes the preperiod and t the postperiod , and we estimate a linear equation of the following form:1yijt=+dj+xijt+ijt in this 2-period pre post model , the coefficient is the diff - in - diff estimate of the effect of being insured during the medicare waiting and captures a linear time trend in yijt . we also expand the specification beyond the simple 2-period pre-/postcomparison to allow multiple pre- and postperiods , given the fact that the health effects may not become apparent in a narrow time frame . taking into account the fact that the shape of the time profile could be different for the di beneficiaries with and without insurance in the waiting period , we interact a flexible function of t with the group indicator dj in order to allow the slopes of the time profile to differ before and after di entry and across groups.2yijt=dj+ftdj+ft+xijt+ijt we model f(t ) as a sequence of dummy variables for each period t. if we set the reference period to the first postperiod , then the coefficient continues to be the diff - in - diff estimate . an attraction of the first - differenced model shown in equations ( 1 ) and ( 2 ) is that permanent unobservable differences between the 2 comparison groups are differenced out . this is desirable in light of some preexisting differences between the 2 groups shown in table 1 . however , the existence of some preperiod differences also suggests that time - varying processes may differentially impact the 2 groups . for example , health shocks might differentially impact the insured di recipients in the waiting period given their poorer initial health . one way of testing for the presence of confounders is to estimate models like those in equations ( 1 ) and ( 2 ) for each potential confounder . this is similar in spirit to the test offered in a previous study in the context of regression discontinuity designs . table 3 shows diff - in - diff estimates based on equation ( 1 ) for 25 potential confounders , including financial variables , health measures , and health - care utilization measures . statistically significant estimates are evidence that the insured di beneficiaries experienced differential pre-/post - di changes in a given variable . there is little evidence of differential impacts for pre-/post - di changes in most of the variables tested , with the exception of a couple of financial variables such as capital income and income from unemployment insurance and workers compensation ( both statistically significant at 10% level ) . to control for these important time - varying processes , we include these variables in first differences in the vector xijt in equations ( 1 ) and ( 2 ) . the study uses 10 available waves of the hrs , which cover the 1992 to 2010 period , with some interviews conducted in 2011 . the hrs is a biennial national representative survey and it interviews individuals born between 1931 and 1941 and their spouses , as well as additional cohorts that have been added in recent waves of the study . the data provide extensive information on health status , employment history , wealth , income , family structure , and government program participation and transfers for a total of 30 672 respondents . for our analysis , it is crucial to accurately line up the dates pre-/post - di program for disability beneficiaries and identify the medicare waiting period . one is entitled to di benefits 5 months after he becomes technically disabled according to the social security definition of disability assuming he has become di insured by accumulating enough work credits . he is entitled to medicare coverage 24 months after his entitlement to di benefits . to identify the medicare waiting period , we must determine the date 1 is entitled to di benefits , which could be before the receipt of the first di paycheck . in other words , this is because it can sometimes take many months or even years before a di application is approved . the processing times are even longer for claims that are initially denied and then subsequently allowed during the appeal process . also , those who are eventually allowed benefits might not file a disability claim immediately following the disability onset . hence , by the time di beneficiaries are notified that their claim has been allowed , some have completed all or part of the medicare waiting period . linking the hrs files to the social security master beneficiary record file , we are able to identify the official date of disability onset . the linkage of hrs and master beneficiary files is restricted and available from the university michigan hrs for researchers who meet the criteria for access to confidential data . knowing the date of disability onset , we can count 5 months from that date to determine the date for di entitlement , which is when the medicare waiting period starts . the waiting period ends 29 months after the date of disability onset . in the hrs , that is , a 2-year passing is between the adjacent interview waves in the hrs . comparing the interview dates with the di entitlement date , we designate the wave right prior to the di entitlement the pre - di period and the wave right after the di entitlement the post - di period . we exclude in the sample the respondents for whom we do not observe their pre - di period and those for whom we do not observe the post - di period . so we include in the sample the individuals who are awarded di benefits , for whom we can observe both the wave before and the wave after their di entry ( waves identified based on the interview date and the di entitlement date if available ) , and for whom we can determine whether they have public health insurance coverage in the wave after di entry . after applying all the sample restrictions , we are left with a sample of 465 respondents . in the 2-period model aka equation ( 1 ) , each respondent is observed twice in the wave before entering di and the wave after . in our analysis , we also expand the specification beyond the 2-period pre-/postcomparison to allow multiple pre- and postperiods aka equation ( 2 ) , given the fact that the health effects may not become apparent in a narrow time frame . in the multiperiod model , each respondent contribute up to 4 observations in the waves pre - di entry and up to 4 observations in the waves post - di entry . our goal is to identify the effects of being uninsured during the medicare waiting period for the newly entitled disability beneficiaries . we distinguish the following 2 groups in our analysis : the newly entitled di beneficiaries who do not have any health insurance coverage before medicare becomes available ( 23% of the sample ) and the newly entitled di beneficiaries who have access to alternative public health insurance ( primarily medicaid ) immediately after entering the di program ( 77% of the sample ) . the former group includes the individuals who lose their health insurance after entering the di program ( 14% ) and the individuals who already lose health insurance prior to the di entry ( 9% ) . among the latter , when we drop this latter group from our study sample , the diff - in - diff estimates became slightly larger in magnitude and stayed statistically significant . the di beneficiaries with health insurance in the waiting period include the individuals who gain access to public health insurance once entering the di program ( 57% ) and the individuals who get their public health insurance from before entering the di program ( 19% ) . as we will show later , the 2 groups appear comparable in a large array of socioeconomic characteristics and health variables at the baseline period . the di beneficiaries who have low income and assets may concurrently receive supplemental security income ( ssi ) benefits . most of the di - ssi concurrent beneficiaries gain immediate access to medicaid coverage because of their entitlement to ssi benefits . the 2 public disability transfer programs are administered by the ssa and follow the same criteria for determining disability severity . hence , the beneficiaries under the 2 programs are comparable at the baseline in terms of their disability / health status , a key variable in our analysis . table 1 presents preperiod descriptive statistics for the sample of newly entitled di beneficiaries without health insurance coverage and those with alternative public health insurance upon entering the di program . the disability beneficiaries with alternative public health insurance during medicare waiting period include more women , fewer blacks , and more married . they appear a bit less healthy than those beneficiaries with health insurance in the waiting period . the former report more health conditions and more difficulties in adls and instrumental activities of daily living . the major differences between the 2 groups lie in some of the financial variables . the disability beneficiaries with insurance in the waiting period , compared to those without insurance in the waiting period , on average earns 40% less from work before entering the di program . however , the former receives nonlabor income ( most likely government transfers ) that is 4.5 times the amount received by the latter . these differentials are not surprising as the uninsured beneficiaries consist of disabled individuals who receive di benefits only ( rather than di - ssi concurrently ) and who are most likely workers with longer worker histories and higher labor earnings compared to the insured disabled individuals who are concurrently receiving the ssi benefits that are means tested . sample means / proportions as of the wave prior to di entry for the di beneficiaries with and without health insurance in the waiting period . abbreviations : adl , activities of daily living ; bmi , body mass index ; di , disability insurance ; iadl , instrumental activities of daily living ; prev , previous ; unemp ins and workers comp , unemployment insurance and workers compensation . net worth is the sum of assets ( primary residence , other real estate , vehicles , businesses , iras , stocks , bonds , checking accounts , cds , and other assets ) less liabilities ( mortgages , other home loans , and other debt ) . major health conditions include ( 1 ) high blood pressure or hypertension ; ( 2 ) diabetes or high blood sugar ; ( 3 ) cancer or a malignant tumor or any kind except skin cancer ; ( 4 ) chronic lung disease except asthma such as chronic bronchitis or emphysema ; ( 5 ) heart attack , coronary heart disease , angina , congestive heart failure , or other heart problems ; ( 6 ) stroke or transient ischemic attack ; ( 7 ) emotional , nervous , or psychiatric problems ; and ( 8) arthritis or rheumatism . what is worth noting is that half of the disability beneficiaries have an annual household total income of less than us$30 000 . it suggests that many beneficiaries could benefit from the recently passed health - care reform , the affordable care act ( aca ) , which makes health insurance more affordable by providing subsidies for families with income below 400% of the federal poverty line to purchase insurance through new health insurance exchanges . the underlying theoretical framework for our research design is a health production function in the 1972 grossman model . individuals inherit an initial amount of this stock that depreciates with age and can be increased by investment . changes in health stock over time are determined by increased health stock less health depreciation . health stock is produced by health inputs such as medical care , health knowledge , lifestyle , environment , and so on . so in our reduced - form estimation about health changes over time ( pre-/post - di ) , we control for changes in medical care ( health insurance coverage and health - care utilization ) , changes in health knowledge ( proxied by years of schooling , an efficiency parameter in the grossman health production function ) , changes in wealth and income , and other socioeconomic variables as proxy for other factors that may affect health outcomes and hard to measure empirically such as lifestyle . in a quasiexperiment research design , we use a diff - in - diff estimator , where the first difference is between outcome variables before and after entering the di program and the second difference is between the disability beneficiaries with health insurance and those without health insurance in the waiting period . taking advantage of the panel data , we take the within - person change in outcome variables before and after entering the di program . this is desirable as it guarantees comparability of the groups in the pre- and postperiods , controlling for any compositional changes due to attrition or sampling variation . one identifying assumption in diff - in - diff models is that other unobserved time - varying processes would have similarly impacted the 2 comparison groups . the preperiod differences in some measures between the groups in table 1 suggest a potential vulnerability on this front . we address this by testing for differential changes in a number of potential confounders , including health measures , financial variables , and health service utilization . we then present in addition to unadjusted diff - in - diff estimates , the estimates that account for within - person changes in these and other relevant variables . let yijt denote the change in health and economic outcomes for individual i in group j = 1 , 0 ( di beneficiaries with and without insurance in the waiting period ) between time t and t 2 ( 2 years between waves in hrs ) . dj is an indicator for being a di beneficiary with insurance in the waiting period . we begin with a 2-period model in which t 2 denotes the preperiod and t the postperiod , and we estimate a linear equation of the following form:1yijt=+dj+xijt+ijt in this 2-period pre post model , the coefficient is the diff - in - diff estimate of the effect of being insured during the medicare waiting and captures a linear time trend in yijt . we also expand the specification beyond the simple 2-period pre-/postcomparison to allow multiple pre- and postperiods , given the fact that the health effects may not become apparent in a narrow time frame . taking into account the fact that the shape of the time profile could be different for the di beneficiaries with and without insurance in the waiting period , we interact a flexible function of t with the group indicator dj in order to allow the slopes of the time profile to differ before and after di entry and across groups.2yijt=dj+ftdj+ft+xijt+ijt we model f(t ) as a sequence of dummy variables for each period t. if we set the reference period to the first postperiod , then the coefficient continues to be the diff - in - diff estimate . an attraction of the first - differenced model shown in equations ( 1 ) and ( 2 ) is that permanent unobservable differences between the 2 comparison groups are differenced out . this is desirable in light of some preexisting differences between the 2 groups shown in table 1 . however , the existence of some preperiod differences also suggests that time - varying processes may differentially impact the 2 groups . for example , health shocks might differentially impact the insured di recipients in the waiting period given their poorer initial health . one way of testing for the presence of confounders is to estimate models like those in equations ( 1 ) and ( 2 ) for each potential confounder . this is similar in spirit to the test offered in a previous study in the context of regression discontinuity designs . table 3 shows diff - in - diff estimates based on equation ( 1 ) for 25 potential confounders , including financial variables , health measures , and health - care utilization measures . statistically significant estimates are evidence that the insured di beneficiaries experienced differential pre-/post - di changes in a given variable . there is little evidence of differential impacts for pre-/post - di changes in most of the variables tested , with the exception of a couple of financial variables such as capital income and income from unemployment insurance and workers compensation ( both statistically significant at 10% level ) . to control for these important time - varying processes , we include these variables in first differences in the vector xijt in equations ( 1 ) and ( 2 ) . in table 2 , we first present unadjusted diff - in - diff estimates of on several health outcome measures ( self - reported poor health , self - reported excellent health , and summary of functional limitations in adl ) and out - of - pocket medical expenditures , and then in table 4 we turn to diff - in - diff estimates adjusted by a large array of socioeconomic variables . unadjusted estimates of health and economic outcomes before and after entering the di program for the di beneficiaries with and without health insurance in the waiting period . column 4 is mean within - individual after - minus - before change in the outcome variable . the new disability beneficiaries with access to public health insurance are 2.5 percentage points less likely to report poor health , compared to before entering di , while we observe an increase of 12.1 percentage points in poor health reporting from the new disability recipients who are uninsured . the unadjusted diff - in - diff estimate of , that is , the effect of being insured in the waiting period , on this health measure is 0.145 , indicating a 14.5 percentage point drop in reporting poor health after entering the di program among those with alternative public health insurance relative to those without . this unconditional effect is statistically significant and the calculations using across group changes and using within - person changes provide similar results ( table 2 , panel 1 ) . the rate of reporting excellent health dropped right after the di entry for both the di beneficiaries with and without health insurance in the waiting period . the proportion reporting excellent health decreased by about half ( from 3.6% to 1.7% ) for the new di awardees with access to public health insurance , while that proportion declined much more dramatically from 8.3% to less than 1% for the uninsured disability beneficiaries during the medicare waiting period . the unadjusted diff - in - diff estimate of the effect of having health insurance in the waiting period on this health measure is 0.055 and statistically significant , indicating a 5.5 percentage point larger drop in reporting excellent health after entering the di program among those without alternative public health insurance relative to those with insurance ( table 2 , panel 2 ) . respondents in the hrs were asked whether they have any difficulty with adl , such as dressing , bathing , walking across a room , eating , getting in and out of bed , and using the toilet . the total number of adls where respondents report any difficulty was close to 1 on average for those insured di beneficiaries before entering di and dropped 8% after entering di . in contrast , the di beneficiaries who are uninsured in the waiting period reported a remarkable increase of about 90% more difficulties in adls when they are uninsured and waiting for the entitlement to medicare . the diff - in - diff estimate on this health measure ( 0.456 ) indicates a statistically significant ( unadjusted ) effect on reducing functional limitations of having alternative health insurance during medicare waiting period ( table 2 , panel 3 ) . the above diff - in - diff estimates on health outcomes indicate that the health status improves or at least does not deteriorate as much for the insured di beneficiaries in the wave post - di compared to the pre - di period . the effect is somewhat surprising : the insured di beneficiaries consist of mainly ssi recipients who , due to the means - tested nature of the program , are in worse health to begin with ( compared to the uninsured di beneficiaries ) because of likely lower investment in health through their life , but they see more improvement ( or less deterioration ) in their health in the waiting period . this evidence likely suggests the positive effect of having alternative health insurance for di beneficiaries in the waiting period . as shown earlier in table 3 , except the different health insurance coverage , there is no differential change pre-/post - di between the 2 groups other than in a few financial variables . the worsening of the few financial variables for the insured beneficiaries post - di is likely to have negative or at least nonpositive impact on their health . but we observe overall advantageous health changes among them compared to the uninsured di beneficiaries . the evidence likely underscores the important role played by having access to health insurance in positively affecting health outcomes during the medicare waiting period . given the fact that these disabled workers are usually forced to drop out of the labor force due to the onset of severe work limitation , they not only lose labor earnings but also likely lose employer provided health insurance . for these workers who have catastrophic health problems , health insurance coverage would be valuable . the 2-year medicare waiting period creates additional barrier for the disabled workers who do not have alternative health insurance coverage during the transition period . the evidence we show may suggest negative health effects of discontinuity in health insurance coverage for individuals with serious health impairments . the punitive effect of the lack of health insurance or access to health care would likely be even larger when we account for the disabled beneficiaries who die while waiting for medicare entitlement . about 2% of the sample dies after entering the di program and before being entitled to medicare . this effect needs further investigation when a larger sample for the disabled beneficiaries becomes available . tests for differential pre-/postchanges in potential confounders for di beneficiaries with and without health insurance in the waiting period . abbreviations : bmi , body mass index ; di , disability insurance ; gov , government ; hrs , health and retirement study ; iadl , instrumental activities of daily living ; psych , psychiatric ; unemp ins and workers comp , unemployment insurance and workers compensation . columns are separate regressions based on equation ( 1 ) for first - differenced dependent variable listed in column head , where differences are taken between the wave just after di entry and the wave just before di entry . d is a dummy for being a di beneficiary with insurance in the waiting period and thus its coefficient measures the relative change in the dependent variable for the di beneficiaries with and without insurance in the waiting period . we also examine the effect on the out - of - pocket medical expenditures of the medicare waiting period . the medical expenditures in table 2 ( panel 4 ) are in the form of the natural log . we see an increase of about 16% in the out - of - pocket medical expenses for those who are uninsured while they wait for medicare to become available , while the di beneficiaries who have alternative insurance during medicare waiting period lower their out - of - pocket medical expenditures by about 14% . the diff - in - diff estimate of the insurance effect is 0.30 , indicating a 30% decrease in out - of - pocket medical expenditures after entering the di program for those with alternative public health insurance relative to those without . in addition , we test the effect of being insured during the medicare waiting period on some other health outcome measure such as self - reported work limitation and the effect on the employment rate . we find the effects statistically significant and with a sign consistent with the hypothesis . however , these unadjusted effects are either primarily driven by the preexisting difference between the 2 groups ( table a1 , panel 1 ) or mainly attributable to the changes in 1 group ( the uninsured di beneficiaries ; table a1 , panel 2 ) . table 4 presents several ols specifications of equation ( 1 ) , which contrasts pre-/post - di changes in health and economic outcomes for the di beneficiaries with and without insurance in the waiting period . the estimation sample has 1 first - differenced observation per respondent . to illustrate the impact of controlling for the potential confounders shown in table 3 column 1 shows the unadjusted diff - in - diff estimate of from table 2 for reference . in column 2 , we add first - differenced demographic variables ( household size and an indicator for being married ) . neither of the additional controls have statistically significant effect on any of the outcomes . in column 3 , we add first - differenced financial variables ( total wealth , net worth , capital income , total nonlabor income , private pension income , income from unemployment insurance and workers compensation , other government transfer income , and other income ) . the diff - in - diff estimate of declines slightly in magnitude for the 3 health outcome measures while it goes up a bit in magnitude for the out - of - pocket medical expenditures . among the financial control variables , statistically significant are only the effect of the total wealth on health reporting and the effect of the government transfer income on the out - of - pocket expenditures , indicating that more wealth is associated with more reports of excellent health and less reports of poor health , and receiving government transfer income here is likely associated with receiving medicaid coverage and then lowering self - paid medical expenses . in column 4 , we add an extensive set of first - differenced health controls that are listed in tables 1 and 4 . again , the diff - in - diff estimates of do not change much for all the outcome measures . among the health control variables , the effects of the onset of cancer , lung disease , heart disease , or high blood pressure on self - reported health status are statistically significant . the onset of high blood pressure or psychiatric problems is shown to have a statistically significant effect on reporting adls difficulties . the onset of high blood pressure , cancer , or stroke has a statistically significant effect on the out - of - pocket medical expenditures . in column 5 , we use multiple pre- and postperiods as shown in equation ( 2 ) , and the estimates of are not very different . in all these first - differenced specifications , we calculate the standard errors allowing for arbitrary correlation in the outcome measures within each of the 2 comparison groups in a given year . for the 2-period models shown in columns 1 to 4 , we use calendar years and obtain 20 group - calendar year clusters . in the multiperiod model shown in column 5 , we cluster by group and time period relative to di entry , which gives 16 clusters ( = 8 periods 2 groups ) . adjusted estimates of health and economic outcomes before and after entering di program for the di beneficiaries with and without health insurance in the waiting period . columns 1 to 4 are separate regressions based on equation ( 1 ) for first - differenced dependent variable , where differences are taken between the wave just after di entry and the wave just before di entry . column 5 is based on equation ( 2 ) for first - differenced dependent variable , where wave - to - wave differences are taken across 4 waves prior to di entry and 4 waves after di entry . d is a dummy for being a di beneficiary with health insurance in the waiting period . income variables include capital income , total nonlabor income , private pension income , income from unemployment insurance and workers compensation , other government transfer income , and other income . we include in the estimation an indicator for missing any health information and an indicator for missing any financial information . standard errors ( in parentheses ) for 2-period models shown in columns 1 to 4 are clustered by group and calendar year ( 20 clusters ) , while standard errors for multiperiod model in column 5 are clustered by group and time period relative to di entry ( 16 clusters ) . the di beneficiaries are primarily workers who lose earnings capacity due to severe health limitations . many lose employer provided health insurance when they are struck by catastrophic health shock and have to leave their jobs . the 2-year waiting period for medicare access creates a health insurance gap and great hardship for them when they are having both negative health shocks and financial shocks and when they need affordable health insurance package more than ever . eliminating the medicare waiting period for disabled workers has been discussed among policy makers and researchers . existing studies on the issue have mainly focused on the fiscal impact of the policy . this study is the first to use a longitudinal panel data set , examine a rich set of controls , and analyze the health and economic consequences of the waiting period for uninsured disability beneficiaries . we take advantage of the longitudinal hrs , which covers the period 1992 to 2010 and provides rich information on health status , employment history , wealth , income , family structure , and government program participation and transfers . linking the hrs files with the social security master beneficiary record file , we are able to accurately line up the timing of pre-/post - di entitlement , which is crucial for our study . in the quasiexperimental research design , we use a diff - in - diff estimator , where the first difference is between the health and economic outcome variables before and after a respondent is entitled to di benefits , and the second difference is between the newly entitled di beneficiaries with access to alternative public health insurance and those being uninsured while waiting for medicare . the adjusted diff - in - diff estimates imply a 13.6 percentage point drop in the proportion reporting poor health , a 6.3 percentage point rise in the proportion reporting excellent health , a drop of total adl limitations , and a 30.5% decline in out - of - pocket medical expenditures among the di beneficiaries with health insurance access relative to those without while waiting for the medicare entitlement . the findings highlight adverse health and economic effects of the lack of health insurance in the medicare waiting period for the uninsured disability beneficiaries . the study has its limitations and we should take caution when we interpret the results . first , in this study , we hope to estimate the effects of having immediate medicare coverage for the newly awarded di beneficiaries without the 2-year waiting period , that is , the counterfactual effects for di beneficiaries if we eliminate the waiting period . to that end , we examine a group of newly awarded di beneficiaries who have alternative public health insurance ( mainly medicaid ) in the waiting period and study the changes in their health and economic outcomes . one assumption for applying this research design is that medicaid and medicare are similar coverage , for example , with similar health care services and similar financing arrangements for individuals . but if medicaid has different effects than medicare for di beneficiaries , our estimation using medicaid insured beneficiaries as a counterfactual would likely produce biased results for the medicare effects on di beneficiaries . second , the 2 comparison groups , the di beneficiaries with and without health insurance in the medicare waiting period , are similar on demographic variables but do not look quite the same in terms of health status at the baseline ( the wave prior to di entry ) . the insured di beneficiaries appear sicker than the uninsured beneficiaries just before entering the di program . to make the 2 groups more comparable at the baseline this , however , would be difficult to implement , given the small sample size in the study . with the differences between the 2 groups at the baseline , there may be alternative interpretations of the results . for example , the health of the insured di beneficiaries does not deteriorate as much or even improve compared to the uninsured di beneficiaries because the former group s initial poorer health may reflect transitory negative shocks , or because the latter group s health may be already on an accelerating downhill trajectory . but these hypotheses are hard to test empirically . third , the 2-year spacing between the hrs waves is not ideal although the hrs is the best available data set for this research . with the 2-year gap between interview waves , it is possible that some of the post - di observations will have become entitled to medicare coverage , even among the designated uninsured group . however , this data limitation unlikely undermines our results and even possibly strengthens our findings . group was clear of those observations who were actually insured with medicare , the differential effects in the health and economic outcomes between the insured and uninsured di beneficiaries would have been even larger than what we estimated . the recently passed aca does not directly address the medicare 2-year waiting period associated with the di program . the act does make some changes to help relieve the problems of this health insurance gap . for example , many di beneficiaries can now join a high - risk insurance pool that were created under the aca and receive immediate insurance coverage while they continue to wait for medicare benefits to become available . in addition , starting in 2014 disabled individuals in this waiting period will have access to expanded insurance options through state - based insurance exchanges and expanded medicaid eligibility . although these new options would not completely solve the problems associated with the medicare waiting period , based on the evidence provided in this article , these policy changes will likely provide at least temporary relief for many affected individuals with disabilities . of course long - term effects of these policy changes in the aca for disability beneficiaries will take time to observe . for example , it is possible that expanded insurance coverage possibilities for disability beneficiaries makes the medicare waiting period less of a deterrence and make di more attractive and thus induced more applications to the di program . on the other hand , more accessible health insurance options in general under the aca will possibly make the di program less appealing to some disabled individuals for whom the main driver to apply to di under the status quo is to get medicare coverage . to understand the impact of the aca reforms on the di program , these are interesting and important aspects to study , especially as the relevant data become available with time passing .
patients with chest pain have a large impact on available resources in coronary emergency rooms ( cer ) . clinical judgement , ecg , risk scores and biomarkers guide in risk stratification . we investigated if high - sensitivity troponin t ( hst ) and the heart score could contribute to risk stratification at the cer . all patients with chest pain , without elevated conventional troponin levels at presentation , were included . hst levels were determined at admission ( t1 ) , at 46 h ( t2 ) and 810 h after symptom onset ( t3 ) . the heart score was calculated as risk score for the occurrence of a major adverse cardiac event ( mace ) . thirty days after discharge , occurrence of mace was registered . eighty - nine patients were included ( overall mean age 61 years ( range 2090 ) ) . at presentation , 68 patients ( 76 % ) had a hst below cut - off value of 14 ng / l ( mean heart score 3.7 , range 19 ) . thirty - one of these 68 patients had a heart score between 13 , no mace occurred in this group . for 3 patients ( 4 % ) hst levels increased above 14 ng / l . these 3 patients had a heart score between 46 . the majority of patients with chest pain can be safely discharged within 46 h after onset of symptoms using hst and the heart score . in contrast , patients with initially normal hst but a high heart score need longer follow - up and repeat hst determination .	an increasing number of patients with chest pain are referred to the casualty department or the coronary emergency room ( cer ) to prove or exclude a potentially serious disease such as an acute coronary syndrome . the challenge lies in rapidly identifying patients who can safely be discharged from the cer . in addition , it is necessary to identify patients who need an aggressive treatment , as part of an acute coronary syndrome ( acs ) , and to take appropriate action as soon as possible . the diagnosis of chest pain is made for 47 % of all patients who are admitted to the cardiology department . acs is not responsible for symptoms in 40 % of patients presenting with chest pain in the cer . the average period of stay of these patients at the cer in our hospital is currently 11 h. given the increasing pressure on the capacity of beds , a shorter length of stay is warranted . the use of biomarkers in addition to risk scores can be helpful in optimising the discharge policy in the cer , through which a shorter duration of stay might be achieved . this study was designed to investigate and evaluate the opportunities for early discharge created by the availability of new and more sensitive biomarkers such as high - sensitivity troponin t assays ( hst ) [ 24 ] . our study focuses on the extent to which the hst in combination with a risk score contributes to the optimisation of earlier decision - making and discharge policy ( 46 h ) in the cer . this study was designed as a prospective cohort study at the cer of the rijnstate hospital in arnhem in the period from february 2011 to july 2011 . the study was approved by the local ethics committee and 131 patients initially participated . the clinical cardiologist and the patients for this study , all patients presenting to the cer with chest pain were eligible . patients could be enrolled if the symptoms had been present for less than 6 h. excluded were patients with an indication for percutaneous coronary intervention ( pci ) , as were patients with st - elevation myocardial infarction ( stemi ) or non - st - elevation myocardial infarction ( nstemi ; typical angina with elevated conventional troponin t ) diagnosed at admission . finally , patients who had been treated for a cardiovascular complication ( e.g. heart failure , unstable angina or myocardial infarction ) in the past month were also excluded . all patients were diagnosed and treated based on conventional troponin t. according to the discretion of the cardiologist , non - invasive ischaemia detection could be performed after discharge , during follow - up . myocardial infarction was defined as a rise and/or fall of conventional troponin with at least 1 value above 30 ng / l . patients presenting to the cer underwent an initial clinical evaluation and , as well as the routine laboratory tests including determination of the conventional troponin t , an additional sample was taken to determine the hst ( t1 ) . this was repeated at 46 h ( t2 ) and 810 h after symptom onset ( t3 ) . if patients presented to the cer within the window of t2 , only the t2 sample was taken . as with conventional troponin t , hst was measured on the e170 module of the modular immunoassay ( roche diagnostics ) . we used 14 ng / l as a cut - off value for hst , above which results were considered to be increased. for the conventional troponin t assay , this value was set at 30 ng / l . the 10 % coefficient of variation is set at 30 ng / l . the coefficient of the variation of 10 % is set at 13 ng / l . the conventional troponin t does not meet the requirement that the coefficient of variation at the 99 percentile limit is below 10 % . the heart score is a scoring system for patients presenting to the cer with chest pain [ 6 , 7 ] . by assigning zero , one or two points to an atypical patient history , ecg abnormalities , the patient s age , any risk factors present , and elevated troponin patients who score 0 - 3 points have < 1 % chance of developing a cardiac event , those scoring 46 points have a 12 % chance , while those patients with a score of 7 have on average a 65 % chance of having a myocardial infarction , percutaneous coronary intervention or coronary artery bypass graft , or death within six weeks after presentation ( mace ) [ 6 , 7 ] . thirty days after discharge , the presence of mace during follow - up was registered retrospectively by checking electronic patient files and personal telephone communication with the patient , with additional information from the general practitioner , when needed . forty - two patients were excluded on basis of the exclusion criteria . finally , 89 patients were included in this study , 52 males and 37 females with an overall mean age of 61 years ( range 2090 years ) . admission time to the cer varied considerably , ranging from 30 min to 6 h after symptom onset . table 2 shows the range and means of the values of hst and conventional troponin t assays measured at the different time points . table 3 shows the results of the combination of hst and the heart score at both t2 and t3 , with the incidence of mace during follow - up . patients with hst < 14 ng / l at t2 and t3 , could have mace during follow - up , whereas no patients with a heart score 3 had mace during follow-up.table 1baseline characteristicsn = 89gender52 male , 37 femaleagemean 61 year ( range 2090)duration of symptomsmean 196 min ( range 30360)heart score ( using ctnt)mean 4.15 ( range 18)history of vascular disease39 ( 44 % ) diabetes mellitus17 ( 19 % ) smoking25 ( 28 % ) hypertension29 ( 32 % ) family history of vascular disease30 ( 34 % ) hypercholesterolaemia32 ( 36 % ) table 2descriptive values of the conventional troponin t and the hst assaysconventional troponin ( ng / l)nminimummaximummeansdt1 ( entry)8916.030.029.61.9t2 ( 4 h 6 h)8911.0333.032.932.3t3 ( 8 h 10 h)8913.0661.041.575.0hst ( ng / l)nminimummaximummeansdt1 ( entry)893.071.910.412.4t2 ( 4 h 6 h)893.0347.014.737.9t3 ( 8 h 10 h)893.0675.424.483.8table 3combining hst with heart score at t2 and t3 in order to calculate the risk for macehst ( t2)nheart scoremaceno mace 14 ng / l3113031(n = 68)3446331371003 14 ng / l01300(n = 21)1346310871035hst ( t3)nheart scoremaceno mace 14 ng / l3113031(n = 65)3146229371003 14 ng / l01300(n = 24)1646412871035 baseline characteristics descriptive values of the conventional troponin t and the hst assays combining hst with heart score at t2 and t3 in order to calculate the risk for mace sixty - eight patients out of 89 ( 76 % ) presented with hst values below 14 ng / l . thirty - one of these patients ( 46 % ) had a heart score between 13 . in this group thirty - seven patients ( 42 % ) with hst levels < 14 ng / l at t2 had a heart score above 3 . in this group , 3 mace occurred in the one - month follow - up ( 8 % ) . these 3 patients all had a pci because of significant coronary stenosis ( angiographically > 70 % and/or fractional flow reserve < 0.8 ) during 30 days of follow - up . in 21 patients ( 24 % ) the probability of a cardiac event in this group was substantially higher ( 28 % ) . in this group , there were no patients with a low heart score . in fig . 1 hst levels at t2 and t3 are compared . at t2 ( 46 h after onset of symptoms ) , 68 patients had a hst value less than 14 ng / l . at t3 ( 810 h after start of symptoms ) , for 3 patients ( 4 % ) hst levels increased to levels above the cut - off value of 14 ng / l ( average increase in these 3 patients was 30 % ) . all 3 patients had a moderately increased heart score between 4 and 6 . based on their heart score , they need longer follow - up and repeat hst determination . one patient had occurrence of a mace during follow-up.fig 1eps changes in serum high sensitivity troponin t ( hst ) in patients with a level 14 ng / l at 4 - 6 h after onset of symptoms eps changes in serum high sensitivity troponin t ( hst ) in patients with a level 14 ng / l at 4 - 6 h after onset of symptoms we investigated the value of the heart score as risk stratification model , with the use of conventional troponin t or hst in patients with suspected acs . our main findings were : patients with normal hst and a heart score of 3 could safely be discharged at t2 , as no mace occurred . with a heart score of 4 or more , further monitoring , serial troponin tests , and/or extra investigation ( exercise testing , nuclear imaging , stress echocardiography ) may be required , because a small proportion of patients have mace at follow - up . patients with elevated hst at t2 and t3 , and/or with a heart score 7 are at high risk of mace . the heart score is a specially validated risk score for patients with chest pain presenting to an emergency department in the netherlands [ 6 , 7 ] . other risk scores such as the grace or timi risk score are mainly validated in patients with stemi and nstemi and therefore less useful for this category of patients [ 8 , 9 ] . it should be noted that these risk scores include elevated cardiac markers in their point scoring system . the heart score system makes use of conventional troponin , and this score system should be further validated , especially with the current widespread use of hst in cardiology practice . in our present study , 3 patients ( 4 % ) with initially normal hst at t2 showed a rise above the cut - off value of 14 ng / l at t3 . the significance of such an increase is at present unclear , as some advocate that a 100 % rise is associated with acute , thrombotic , myocardial infarction . if clinical decision - making had been based on hst alone , these 3 patients would not be identified as high - risk patients at risk for mace . by also taking the heart score into account the use of a risk score as the heart score can provide additional information for identifying these patients . as mentioned earlier , the heart score has been validated for conventional troponin t and not for hst . it is to be expected that by using a high - sensitivity assay in a new heart score , hst levels will more often be elevated as compared with conventional troponin t , and hereby the new risk score could have a higher sensitivity , at the cost of less specificity . further studies should provide conclusions regarding the discriminative value of this new risk score . despite the fact that cardiac troponins are sensitive and are specific biochemical markers of myocardial damage , an increased troponin t level occurs in many diseases in which the heart does not show ischaemia , such as congestive heart failure , pulmonary embolism , renal failure , acute neurological disease , myocarditis or sometimes in apparently healthy persons . furthermore , the availability of new high - sensitive assays for troponin t may allow the detection of small changes in troponin and this test meets the requirements of the universal definition of myocardial infarction . the universal definition of acute myocardial infarct states that there has to be a typical rise and/or fall of biochemical markers ( preferably troponin ) with at least 1 value above the 99 percentile reference limit with at least one of the following : ischaemic symptoms , ecg changes , pathological q waves or imaging evidence of new loss of viable myocardium motion abnormality . the hst assay does meet the requirement that the coefficient of variation at the 99 percentile limit is below 10 % . the test does not only appear more sensitive , but is able , even 3 h after symptom onset , to differentiate whether there is an acute coronary syndrome or not . early decision - making is therefore possible . a problem that may arise is the possibility of false - positive results of acute coronary syndrome . however , it is still unclear whether there should be an absolute or relative increase in troponin values . in addition , at this moment it is also unclear how much the troponin values should increase to speak about an acute coronary syndrome . some advocate that a doubling of the serum hst within 3 h indicates an acute coronary syndrome whereas others ( including the guidelines ) state that there has to be a rise and/or fall with at least 1 value above 14 ng our study is relatively small ; therefore , future studies should be aimed at extending these findings by including more patients . only with evidence from larger study populations , conclusions for clinical decision - making can be drawn . , our study suggests that patients with chest pain at the cer , with a negative hst level together with a low heart score , can be safely discharged from the hospital within 4 - 6 h after onset of symptoms .
objectives . this study is a questionnaire survey on the use of pharmacological agents to modify liver ischaemia - reperfusion ( ir ) injury in patients undergoing hepatectomy for colorectal liver metastases with the target population being those units participating in the livermetsurvey international registry . methods . members of livermetsurvey were sent an online questionnaire using surveymonkey comprising ten questions on the use of pharmacological agents to modulate hepatic ir injury in the perioperative period after hepatectomy . the questionnaire was sent to 446 clinicians registered with the livermetsurvey . there were 83 ( 19% ) respondents . results . fifty - two ( 77% of 68 respondents to this question ) never used pharmacological agents to modify liver ir injury during hepatectomy . thirteen ( 19% ) used pharmacological agents selectively . three ( 4% ) used these routinely . n - acetylcysteine was the most widely used pharmacological agent with equal distribution of use around intraoperative and postoperative periods . conclusions . this is believed to be the first survey on the use of pharmacological agents to modify liver ir injury . the target population is clinicians involved in liver resection . the results show that pharmacological modulation is used by only a minority of respondents to this questionnaire and that when this treatment is selected , n - acetylcysteine is the most frequently used .	hepatectomy is established as a standard of care for patients with resectable liver - limited metastases of colorectal cancer origin [ 1 , 2 ] . hepatectomy is also undertaken for a range of other malignant and benign conditions [ 3 , 4 ] . contemporary experience with techniques such as downsizing of tumours by neoadjuvant chemotherapy and modification of the volume of the future remnant liver by selective portal vein embolization results in the ability to offer liver resection to a greater proportion of patients with liver metastases . these changes , coupled with population trends resulting in a greater proportion of elderly patients presenting for treatment , mean that , in the 21st century , liver resection for metastatic colorectal cancer is often undertaken in older individuals and/or in patients whose hepatic functional reserve may have been compromised by prior chemotherapy or by preexisting chronic liver disease . although reports outline the feasibility of undertaking liver resection with low perioperative mortality [ 9 , 10 ] , postoperative morbidity is important as it can influence a range of outcomes including critical care occupancy and in - patient stay , uptake of adjuvant chemotherapy , and quality of life after surgery . during liver resection , ir injury can occur during any of a series of operative steps including liver mobilisation and permanent inflow occlusion of the segment to be resected and during temporary inflow occlusion followed by restoration of blood flow to the future remnant liver ( the pringle manoeuvre ) . ir injury is an important determinant of postoperative morbidity and eventual clinical outcome [ 11 , 12 ] . strategies employed to minimise ir injury include avoidance of inflow occlusion and ischaemic preconditioning and the administration of pharmacologic agents . at the cellular level , kupffer cells release proinflammatory mediators during liver ir , including oxygen - derived free radicals . the physiologic intracellular defence mechanisms involved in the regulation of oxidative injury include enzymatic pathways catalysed by superoxide dismutase and glutathione peroxidase . glutathione ( gsh ) , a tripeptide composed of glycine , glutamic acid , and cysteine , constitutes the largest component of the endogenous thiol buffer for oxygen - derived free radicals . for tissue gsh synthesis , n - acetylcysteine is a thiol containing synthetic compound . in the isolated , perfused rat liver , administration of nac before reperfusion results in concentration - dependent increases in gsh concentrations in bile with corresponding increases in bile flow [ 19 , 20 ] . currently the most established clinical use for n - acetylcysteine is in the treatment of acetaminophen ( paracetamol ) poisoning [ 21 , 22 ] . to date one small clinical trial has assessed the effect of perioperative n - acetylcysteine on prevention of ischemia - reperfusion injury in liver resection . liver function was assessed by postoperative transaminase ( alt ) rise and this was significantly ameliorated in the treatment group . despite the predominantly favourable results of experimental studies there are no substantive clinical trials evaluating the role of n - acetylcysteine or other pharmacological agents to modulate ir injury in patients undergoing hepatectomy . these drugs ( in particular n - acetylcysteine ) have been available for a long time and anecdotal evidence suggests that they are still used in an off - licence mode as modifiers of liver ir in hepatectomy . a questionnaire survey provides an overview of the contemporary use of pharmacological modulation of ir injury in liver surgical practice . an international target population of clinical units undertaking liver surgery can be obtained through the livermetsurvey . thus the aim of this study is to undertake a questionnaire survey of the use of pharmacological agents to modify liver ir injury in patients undergoing hepatectomy for colorectal liver metastases in units participating in the livermetsurvey . the questionnaire design examined strategies for modulation of liver ir injury during hepatectomy and included questions on a broad overview of techniques for this purpose in addition to pharmacological interventions . specific detail was sought on whether pharmacological agents were used by respondents to treat or reduce liver ir injury with information being sought on the indications for use , choice of agent , use of a loading dose , timing of use in relation to the stage of surgery ( preoperative , intraoperative , posttransection , etc . ) , and dose . as it was anticipated from prior literature searches that n - acetylcysteine would be the most widely used agent , specific focus was given to this drug [ 24 , 25 ] . a final question sought opinion on whether the respondent supported in concept a randomized trial to evaluate the role of pharmacological ir modifiers in hepatectomy . permission was obtained from the livermetsurvey for their members to be sent an online questionnaire using surveymonkey ( https://www.surveymonkey.com/ ) . the livermetsurvey was selected as this is a registry of liver resection outcomes for colorectal hepatic metastases and thus all participants would be likely to be undertaking hepatectomy . response to this questionnaire was discretionary and following the initial email no reminders were sent . the questionnaire was live for a period of six weeks from june 18 , 2013 , and was sent to 446 participants of the livermetsurvey . there were 85 respondents of whom two were not actively engaged in liver resection practice . these two respondents were excluded to provide a final study population of 83 ( 19% ) respondents . the study was discussed with the hospital 's research and development team , who advised that , as there was no patient contact and only email contact with professional colleagues , no ethics committee submission was required . the number of respondents to each individual question is provided and serves as the denominator for the percentage of respondents to that particular question . for several questions , more than one response was permitted and therefore the cumulative total of percentage responses to such questions can exceed 100 . there were 72 respondents to this question and more than one response was permitted ( figure 1 ) . the most frequently used strategy was avoidance of the pringle manoeuvre by 53 ( 74% ) respondents . six ( 8% ) replied that they used pharmacological agents to modify liver ir injury . fifty - two ( 77% ) stated that they never used pharmacological agents to modify liver ir injury during hepatectomy . ten ( 56% ) stated that a requirement for total vascular exclusion was an indication for the use of pharmacologic agents , 9 ( 50% ) stated that prolonged inflow occlusion was an indication , 9 ( 50% ) used pharmacological agents if there was evidence of deterioration in liver function in the postoperative period , and 8 ( 44% ) used pharmacological agents in the setting of a small - volume future remnant liver , 7 ( 39% ) in major liver resection of more than 3 couinaud segments , 5 ( 28% ) in liver resections undertaken in patients with cirrhosis , and 4 ( 22% ) in patients who had received prior chemotherapy . individual replies were received ( one respondent each ) for glutamine , desflurane , sevoflurane , bicarbonate , arginine , and a cocktail of methionine with vitamins c and e. there were 22 responses to this question with 14 ( 64% ) replying that they did not use a loading dose . three ( 17% ) used pharmacological intervention prior to surgery in the form of steroids . two ( 11% ) gave nac at induction of anaesthesia , four ( 22% ) during liver mobilisation , two ( 11% ) at commencement of transection , two ( 11% ) postoperatively ( routinely ) , and five ( 28% ) postoperatively ( selectively ) . there were 9 respondents to this from those who used n - acetylcysteine for modulation of liver ir injury . seven ( 78% ) used a standard dose for all patients and 2 ( 22% ) used a body weight - adjusted dose . three ( 33% ) continued the infusion for 48 hours , 3 ( 33% ) until serum transaminases started to fall , 2 ( 22% ) for 72 hours , and 1 ( 11% ) for 24 hours . fifty - six ( 81% ) answered affirmatively and 6 ( 9% ) replied that there should not be a trial . this study reports the results of what is thought to be the first survey of the use of pharmacological agents to modify liver ir injury in the perioperative period . first , the results do not in any way represent an official position statement of the livermetsurvey . second , given that less than 20% of the target audience responded to this questionnaire , the replies are not indicative of any consensus view . third , it is possible that those who responded had a vested interest in pharmacological modification of liver ir injury and thus the results could be skewed by ascertainment bias . the target population was selected as the livermetsurvey participants as by definition these are units undertaking liver surgery for colorectal hepatic metastases . clearly , the views of units and clinicians outwith livermetsurvey will not have been captured by this survey . interpretation of these results bearing these caveats in mind produces an interesting overview into pharmacological modification of liver ir injury . first , only a minority of respondents use pharmacological agents to modify liver ir injury during hepatectomy with 6 ( 8% ) of 72 respondents stating that they used this strategy ( figure 1 ) . the influence of the phrasing of the question on the answer is seen in that when asked specifically whether pharmacological agents were used to modify liver ir injury in the next question , 13 ( 19% of 68 respondents ) replied that they used these agents selectively in addition to the 3 ( 4% ) who used them routinely . nonetheless it is clear from this small study that pharmacological modification of ir injury during hepatectomy is only used by a minority of respondents . despite the negative results of a randomised controlled trial of ischaemic preconditioning to reduce liver ir injury the selection criteria for use of pharmacologic agents are interesting in that those clinicians using these agents employed them for major resections and in individuals with compromised liver parenchyma or function . this observation may be worth bearing in mind if a future randomized trial is to be considered as targeted use may be more appropriate than routine use in all liver resections . in terms of choice of pharmacological agent , it should be emphasised that the questionnaire restricted the responses and that only 18 responses were received to this question with n - acetylcysteine used without a loading dose being the most frequent choice . there is clearly no consensus on the optimum time for commencement of nac or on the duration of use although the majority of respondents favoured a standard dose for all patients without adjustment for body weight . the majority of respondents ( 56 ( 67% of cohort ) ) support a randomised trial and in view of patterns of current use it would appear that the role ( if any ) to be explored is in patients undergoing extended resection or with compromised functional hepatic reserve or with a small remnant liver after resection . in summary , this is thought to be the first questionnaire survey of a population of clinicians undertaking surgery for colorectal liver metastases to investigate the use of pharmacological agents for modification of liver ir injury . although the response rate was only 19% , the resultant population of 83 clinicians is the largest specialist group to contribute data to this question to date . the results show that pharmacological modulation of liver ir injury is not a routine component of care after liver resection in contemporary hepatic surgery practice and that it is used routinely by only 3 ( 4% ) respondents within livermetsurvey and selectively by a further 13 ( 19% ) with the indications being major resections or in patients with compromised liver reserve .
the goal of the present study was to determine if the expression of metallothionein isoform 3 ( mt-3 ) might serve as a biomarker for human bladder cancer . to accomplish this goal , we defined the localization and expression of mt-3 protein and mrna using fresh and archival biopsy specimens obtained from patients undergoing differential diagnosis for a variety of bladder disorders . we used immunohistochemistry , immunoblot , and rt - pcr analysis to define the localization and expression of mt-3 protein and mrna . immunohistochemical analysis disclosed no immunoreactivity for mt-3 in normal bladder cells . the absence of mt-3 expression in the normal bladder was further confirmed by demonstrating that mt-3 mrna could not be detected using reverse transcriptase - polymerase chain reaction ( rt - pcr ) or mt-3 protein using immunoblot . immunohistochemistry also disclosed no immunoreactivity for mt-3 in archival biopsy specimens from patients with interstitial cystitis and related disorders . immunohistochemical analysis demonstrated that mt-3 was expressed in carcinoma in situ ( cis ) , high - grade bladder cancer , low - grade bladder cancer , and dysplastic lesions . mt-3 immunostaining was intense in both cis and high - grade bladder cancer , and low to moderate in low - grade bladder cancer and dysplastic lesions . we determined mt-3 mrna expression in a subset of these bladder cancer specimens ; expression was elevated as compared to that of the housekeeping gene , ss - actin . the cdna from the rt - pcr reaction primed for mt-3 contained a foki restriction site , a site unique for mt-3 as compared to other mt family members . in conclusion , this study demonstrates that mt-3 is up - regulated in human bladder cancer and that this up - regulation increases with increasing tumor grade . the finding that mt-3 expression is minimal in normal bladder suggests that mt-3 might be developed into an effective biomarker for bladder cancer.imagesfigure 1figure 2figure 3figure 4
we present a patient with an animal - type malignant melanoma associated with the nevus of ota in the orbit who showed a good prognosis after a combination of orbital extirpation , chemotherapy , stereotactic radiotherapy , and gamma knife . a 42-year - old japanese woman presented with two tumors , one pathologically diagnosed as right - sided intraconal animal - type malignant melanoma and the other intracranially , presumed to be of the same pathogenesis and both were considered to have arisen from the nevus of ota . she underwent an extirpation of the orbit , chemotherapy ( dav therapy , which is a combination of dacarbazine , nimustine , and vincristine ) , stereotactic radiotherapy ( 54 gy in 27 fractions ) , and gamma knife ( marginal dose was 17 gy , target volume was 0.2 ml ) . she has been alive for 33 months since the extirpation , with no sign of local recurrence , new metastasis , nor enlargement of the intracranial tumor . not just combination therapy but also the low malignancy of animal - type melanoma may have contributed toward the good prognosis .	the nevus of ota is a light bluish nevus on the unilateral face , which is congenital or acquired in adolescents , and often seen in asian females . malignant transformations are rarely observed in the orbit 1 and seldom seen among asians 2 . as the potential for local invasion or metastasis varies widely , strict prognostic indicators for orbital melanoma have yet to be established 3 . animal - type melanoma is a rare type of malignant melanoma that usually arises in skin lesions 4 and shows indolent progression and good prognosis despite metastasis . to the best of our knowledge , this is the first case report of a primary animal - type malignant melanoma in the orbit that developed from the nevus of ota to show a satisfactory clinical course . a 42-year - old woman presented with a right - sided eye swelling and fever . she had been diagnosed with diabetes mellitus 2 years previously , but had received no medication . she had no particular family history , except for the fact that her father had stomach cancer . bluish pigmentations were visible on her right forehead , ear , and eyelid since birth . hertel exophthalmometry showed exophthalmos in her right eye [ 24 and 18 mm in the right and left eyes , respectively , with a base of 110 mm ( fig . ocular movement of her right eye was restricted in both the horizontal and the upward gaze . intraocular pressure was 19 mmhg in her right eye and 18 mmhg in her left eye . mri showed a right - sided exophthalmos and a 2.53 cm large , well - defined intraconal mass . the tumor was heterogeneous and there was a slightly high intensity observed in the gadolinium - enhanced t1-weighted image ( t1-wi ) and a low intensity in the gadolinium - enhanced t2-weighted image ( t2-wi ) ( fig . another 56 mm large mass that was discontinuous with the orbital tumor in the cranial base was also observed and primarily presumed to be a meningioma ( fig . laboratory tests indicated that her diabetes mellitus was uncontrolled ( hba1c , 11.5% ) and that she had a normal ldh level ( 220 u / l ) . ( a ) bluish pigmentation was seen on the right eyelid and black pigmentation was seen in the right conjunctiva . ( b ) t1-weighted image : right - sided exophthalmos was seen and the retrobulbar mass showed a slightly high intensity in the t1-weighted image ( indicated by an arrow ) . ( c ) t2-weighted image : right - sided exophthalmos was seen and the retrobulbar mass showed low intensity in the t2-weighted image ( indicated by an arrow ) . a 56 mm large mass , which was discontinuous with the orbital tumor in the cranial base , was also enhanced ( indicated by an arrowhead ) . the tumor , which ruptured during the operation because of biopsy for pathological diagnosis , was frozen for later analysis . because the subsequent pathological diagnosis of the frozen specimen during the operation showed it to be a benign lesion , we completed tumor excision without orbital extirpation . ( a ) macroscopy of the extracted orbital tumor : highly pigmented 62 cm large tumor , which ruptured during the extirpation . ( b ) hematoxylin and eosin staining of the orbital tumor ( 20 ) : melanin - containing cells ( melanocytes and melanophages ) are abundant in the tumor . ( c ) hematoxylin and eosin staining of the orbital tumor after using a bleaching method for melanins ( 20 ) : diffuse proliferation of the epithelioid cells and spindle cells , and low - grade nuclear atypia were seen . ( e ) melan - a staining ( 20 ) : positive for atypical cells . ( f ) hematoxylin and eosin staining of the orbital tumor after using a bleaching method for melanins ( 20 ) : vast areas of necrosis were seen . after preparation of the sample , the pathological examination of the tumor showed that the lesion was an animal - type malignant melanoma . after using a bleaching method for melanins , hematoxylin and eosin staining was performed ( fig . 2c ) . all the cells had low - grade pleomorphic nuclei , with nucleoli observed in some cells , although they were not found to be prominent . hmb ( human melanoma black)-45 ( fig . 2d ) and melan - a ( fig . we performed orbital extirpation with an excision of a 5 mm margin from the nevus of ota ; the orbit was then covered with a skin graft taken from the patient s thigh . these cells were found in the skin , subcutaneous fat , orbital muscle , orbital fat , lacrimal gland , and sclera . results also showed that the retina , the uvea , and the cornea were not involved . some parts of the nevus were cellular , with slightly large , round epithelioid melanocytes that contained low - grade pleomorphic nuclei . although some of these were accompanied by small nucleoli , neither necrosis nor mitosis was observed . although no malignant changes were apparent at the time of the procedure , the findings did suggest the possibility that these atypical melanocytes might be an early stage of melanoma . mri performed before the second operation showed that there was a slight enlargement and high intensity in the t1-wi of the cerebral mass that had been considered as a meningioma before . in addition to the second surgery , chemotherapy ( dav therapy , which is a combination of dacarbazine , nimustine , and vincristine ) , stereotactic radiotherapy ( 54 gy in 27 fractions ) , and gamma knife ( the marginal dose was 17 gy and the target volume was 0.2 ml ) were administered . since then , we have examined her once every 23 months by laboratory tests including lactate dehydrogenase , aspartate aminotransferase , and alanine aminotransferase . we have also taken some biopsies to check the orbital local recurrence and performed annual 18f - fluorodeoxyglucose positron emission tomography to check metastasis . otolaryngologists and dentists have checked the pigmentation of the mouth and physicians have brought her severe diabetes mellitus under control . thirty - three months have passed since the second surgery , with no sign of local recurrence , new metastasis , nor enlargement of the intracranial tumor . the nevus of ota ( oculodermal melanocytosis , naevus fusculocoeruleus ophthalmomaxillaris ) was described by the japanese dermatologist , ota , in 1939 . it is a light bluish nevus on the unilateral face , which is congenital or acquired in adolescents . it has been shown to be more common in east asians ( 0.21.0% ) than in western populations ( 0.04% ) 5 . the incidence of malignant change is 0.5% for east asians and about 25% for western populations 2 . therefore , the calculated incidence of malignant melanoma associated with the nevus of ota is 0.0010.005% ( 0.21.0%0.5% ) among east asians , whereas it is 0.01% ( 0.0425% ) among western populations . malignant melanoma associated with nevus of ota has been reported to occur in the uveal tract 6 , optic nerve head 7 , brain , and orbit . as for western populations , rice and brown 8 reported finding six cases of primary orbital melanoma associated with nevus of ota , whereas two other studies have additionally reported two more cases 1,9 . however , in terms of east asian populations , ours is the first report of such a case . the original report of the animal - type melanoma was based on a highly pigmented malignant melanoma that was found in a horse and then shown to be a very rare variant of melanoma . it has been reported that this melanoma usually occurs in skin lesions found on the scalp , face , and extremities 10 . of the eight reported orbital melanoma cases , pathological diagnosis indicated that five were spindle , two were epithelioid , and one was mixed 1,8,9 . the current patient is the first case of the animal - type melanoma that arose in the orbit . histopathologically , animal - type melanoma cells show some nuclear atypia and regular oval to round nuclear shapes , with moderate anisonucleosis , small nucleoli , and distributed chromatin . characteristically , these do not fulfill the cytologic criteria of malignancy . the most important clue to the diagnosis of animal - type melanoma is effacement of the dermal architecture by areas of confluent melanocytic growth . all the cells had low - grade pleomorphic nuclei , with nucleoli seen in some cells . distant metastases of animal - type melanoma are rare and even when they occur , these patients appear to have a better prognosis than those with an ordinary melanoma presenting with metastatic disease 10 . it may because of this characteristic that our patient showed neither enlargement of tumor nor neurologic symptoms for long despite an intracranial metastasis . however , even with this low malignant potential , animal - type melanoma is clearly a variant of melanoma 10 . therefore , application of the guidelines used for melanoma , clinical investigation of regional and distant metastases , and surgical treatments with safety margins 12 are recommended in these types of cases 13 . thirty - three months since the extirpation , the patient is still alive , with careful follow - up examinations , despite having intracranial metastasis . not just the combination therapy but also the low malignancy of animal - type melanoma may have contributed toward this good prognosis . although the possibility is not high that malignant transformation occurs in the nevus of ota , it is important to consider this possibility as it can be seen in any population groups . doctors involved in the treatment of nevus of ota ( as in our case , ophthalmologists , pathologists , dermatologists , brain surgeons , otolaryngologists , radiologists , and dentists ) should understand that malignant transformation could occur and should cooperate with one another for its treatment . although practical strict prognostic indicators for orbital melanoma are yet to be established 3 , this particular case suggests that the characteristic indolent progression of the animal - type melanoma might partly contribute toward the good prognosis .
objectives . type 2 diabetes ( t2d ) with ketosis was common because of late diagnosis and lacking adequate treatment in rural regions of china . this study aimed to provide the data of t2d with ketosis among inpatients in a south - west border city of china . methods . data of 371 patients of t2d with ketosis who were hospitalized between january 2011 and july 2015 in baoshan people 's hospital , yunnan , china , were analyzed . new - onset and old - diagnosed t2d patients presenting with ketosis were compared according to clinical characteristics , laboratory results , and chronic diabetic complications . results . overall , the blood glucose control was poor in our study subjects . male predominated in both groups ( male prevalence was 68% in new - onset and 64% in old - diagnosed groups ) . overweight and obesity accounted for 50% in new - onset and 46% in old - diagnosed cases . inducements of ketosis were 13.8% in new - onset and 38.7% in old - diagnosed patients . infections were the first inducements in both groups . the prevalence of chronic complications of diabetes was common in both groups . conclusions . more medical supports were needed for the early detection and adequate treatment of diabetes in rural areas of china .	the prevalence of type 2 diabetes ( t2d ) in rural regions is rising rapidly and has been a great challenge to the local health care programs . in china , large parts of diabetic individuals lived in rural areas , where resources to screen and treat diabetes and its complications were limited [ 13 ] . so far , the situations of type 2 diabetes presenting with ketosis prior to admission in the south - west rural region were unclear . ketosis is a symbol of acute disorder of glucose and lipids metabolism in diabetic patients . ketosis or ketoacidosis has been regarded as trait of type 1 diabetes ( t1d ) . because of backward in economy , quite a few patients did not get duly treatment until severe hyperglycemia or diabetic complications occurred . our present study was to investigate the prevalence and characteristics of t2d presenting with ketosis in rural areas aiming to suggest more efforts for the prevention and control of diabetes in rural areas of china . we performed a retrospective analysis of t2d with ketosis in patients admitted to the endocrinology department of baoshan people 's hospital of yunnan province between january 2011 and july 2015 . overall , data of 391 t2d presenting with ketosis inpatients aged 12 years or older were enrolled . nine patients were excluded because of surgery , pregnancy , trauma , secondary diabetes , or pancreatic exocrine diseases . thus , the other 371 patients ( 245 males and 126 females ) were allocated to the analysis . according to the diabetic duration new - onset group included those with new diagnosed diabetes or no more than 6 months after onset . old - diagnosed group were patients of previously diagnosed t2d presenting with ketosis at this admission . t2d was diagnosed if patients had clinical and metabolic features including overweight or obesity , more than 40 years age at onset , and obvious diabetes family history in combination with preserved -cell function ( fasting c - peptide of more than 0.33 t2d with ketosis was diagnosed if patients met the criteria of t2d and had positive urinary ketone body results this time on admission . urinary ketone body positive was diagnosed if urinary acetoacetate increased over 15 mg / dl by using sodium nitroprusside method . acidosis was diagnosed if an arterial ph of less than 7.35 and/or blood bicarbonate level of less than 15 overweight and obesity were defined if body mass index ( bmi ) 24 , which was set forth by chinese guidance of overweight and obesity in adults . data of demographic and clinical characteristics including age , gender , height , weight , bmi , blood pressure , and diabetic family history were collected . laboratory parameters including plasma glucose , hba1c , c - peptide , total cholesterol , and triglycerides were recorded . the chronic diabetic complications including peripheral neuropathy , retinopathy , diabetic foot , and persistent microalbuminuria that were evaluated by trained physicians during the hospitalization . all of the data were analyzed by jmp 9.0 ( sas institute , cary , nc ) . the anova or mann - whitney test analysis was used to evaluate the differences in continuous variables . table 1 showed the clinical characteristics of the new - onset and old - diagnosed t2d with ketosis patients . among the total 371 subjects , 211 of the patients ( 57% ) were classified as new - onset , while 160 ( 43% ) were old - diagnosed t2d with ketosis . the subjects of new - onset group had an average duration of 1.7 months with weight loss of 7.3 kg , while the old - diagnosed group had an average diabetic duration of about 6.3 years with weight loss of 9.2 kg . subjects of new - onset group were younger ( 47 12 versus 53 13 yr , p = 0.001 ) and more overweight ( 50% versus 46% , p = 0.009 ) than those old - diagnosed t2d with ketosis . family history of diabetes was similar ( p = 0.79 ) between new - onset ( 35% ) and old - diagnosed ( 34% ) group . the blood pressure of the analyzed subjects was not significantly different between two groups . table 2 showed the biochemical characteristics of the new - onset and previously diagnosed t2d patients presenting with ketosis . fasting plasma glucose mmol / l ) and old - diagnosed ( 16.4 7 mmol / l , p = 0.49 ) group . 2 h plasma glucose was also similarly elevated ( p = 0.86 ) between new - onset ( 24.2 8 fasting c - peptide levels were similar ( p = 0.60 ) between new - onset ( 0.69 0.11 nmol / l ) and old - diagnosed ( 0.58 0.17 2 h c - peptide was significantly higher ( p = 0.02 ) in new - onset ( 2.58 1.1 the hemoglobin a1c ( hba1c ) and fructosamine were not different between the two groups . more patients in the old - diagnosed group ( 19.4% ) suffered from acidosis than new - onset group ( 6.6% ) . total cholesterol ( 5.9 1.7 versus 4.9 1.8 mmol / l , resp . , in new - onset and old - diagnosed group , p < 0.0001 ) and triglyceride ( 4.4 4.0 versus 3.7 4.2 mmol / l , resp . , in new - onset and old - diagnosed group , p = 0.004 ) were significantly higher in new - onset than in old - diagnosed group . those new - onset patients showed a lower incidence of predisposing factors than previously diagnosed t2d patients presenting with ketosis ( 13.8% versus 38.7% , p < 0.001 ) . the potential triggers for subjects in t2d with ketosis were varied . among them , infections were predominant in both groups . the prevalence of respiratory system infection was 10.3% in new - onset and 14.9% in old - diagnosed group . urinary system infection was 1.8% in new - onset and 9.9% in old - diagnosed group . figure 2 showed the prevalence of chronic diabetic complications in new - onset and previously diagnosed t2d with ketosis . the patients with new - onset had higher ( p < 0.0001 ) prevalence of fatty liver disease ( 26.2% ) compared with those of old - diagnosed ( 15.1% ) group , while the prevalence of most chronic complications was higher in old - diagnosed group than new - onset group , including retinopathy ( 8.1% versus 17.8% , resp . , in new - onset and old - diagnosed group , p < 0.0001 ) , peripheral neuropathy ( 25.3% versus 47.6% , resp . , in new - onset and old - diagnosed group , p < 0.0001 ) , diabetic foot ( 13.3% versus 23.2% , resp . , in new - onset and old - diagnosed group , p < 0.0001 ) , and persistent microalbuminuria ( 8.2% versus 15.1% , resp . , in new - onset and old - diagnosed group , p < 0.0001 ) . no significant differences ( p = 0.45 ) in atherosclerosis were found in new - onset ( 18.0% ) and old - diagnosed ( 20.1% ) group . in this study , we showed the characteristic of t2d with ketosis in rural areas of south - west part of china . it was concluded from the study that the control of t2d was poor in rural area , as reflected not only by extremely elevated hba1c and plasma glucose performed once on admission but also by high prevalence of chronic diabetic complications evaluated during hospitalization . there were limited data about the prevalence of ketosis in type 2 diabetes population . in a study of 134 chinese patients with severe hyperglycemia , there were 24 patients diagnosed with ketosis ( 17.9% ) and 6 patients diagnosed with ketoacidosis ( 4.5% ) . subjects of new - onset and old - diagnosed t2d with ketosis group had similarities and differences at clinical manifestations . consistent with previous studies , we found a predominance of male proportion in the study subjects [ 711 ] . the underlying mechanisms of male predominance in t2d with ketosis were unknown and needed further investigation . differences of body fat distribution and sex hormone were supposed to be factors contributing to the gender difference [ 12 , 13 ] . in our study , the prevalence of hyperlipidemia was high in both new - onset and old - diagnosed subject , which were true with many previous studies [ 11 , 1416 ] . some investigators have reported that acute elevation of free fatty acid levels in the circulation increased insulin resistance and impaired -cell function [ 17 , 18 ] . in our study subjects , the prevalence of hyperlipidemia was 60% in subjects of new - onset group and 29% in old - diagnosed group . we analyzed our data and found that hyperlipidemia was common in the overweight diabetic subjects . bmi of the enrolled patients positively correlated with both triglyceride and total cholesterol level ( data not shown ) . the phenomenon further confirmed the view that hyperlipidemia played substantially roles underlying trigger of ketosis especially in new - onset diabetes . therefore , interventions for prevention and treatment of overweight and hyperlipidemia were essential in t2d patients . infections were the most common precipitating factors for ketosis in t2d patients , which was an important point distinguishing t2d from t1d . in patients with t1d , noncompliance with insulin therapy was considered as first reason for precipitating ketosis [ 14 , 19 ] . in our old - diagnosed t2d subjects , varied infections accounted for about 40% of the total precipitating factors , indicating that infections were the important precipitating factors leading to ketosis . however , in new - onset ketosis subjects , most cases were nonprovoked ketosis . the discrepancy in prevalence of precipitating factors might indicate the complexity and heterogeneity of t2d with ketosis . more clinical epidemiological surveys need to be conducted to clarify the heterogeneity in t2d with ketosis . in our study subjects , patients had a higher incidence of diabetic chronic complications , especially in those previously diagnosed t2d patients . it was shown by literatures that those who came for regular follow - up diabetic treatment had a lower incidence of retinopathy and nephropathy compared to those who had irregular follow - up . since t2d and most of the chronic complications were silent at first , patients usually did not come to the clinic for routine examination until they developed severe complications of diabetes in rural areas . as a result , most patients were complicated with severe chronic complications at their fist diabetic diagnosis . however , late treatments of diabetes and its complications were often associated with higher costs to the affected patient [ 1 , 20 ] . therefore , it was reasonable to suggest detection and adequate treatment at an earlier stage , by which the excess cost due to treatment of diabetes complications might be minimized . first , we did not perform plasma ketosis test which was more sensitive and specific in detecting ketosis ; however , urinary ketone body measurements were widely acceptable in hospitals for diagnosing ketosis . second , we did not measure the body fat distribution which was a potential and important parameter for explaining gender difference in t2d with ketosis . third , the single - center study restricted our ability to assess the overall characteristics of t2d with ketosis in rural areas . further large cohorts and prospective follow - up studies were needed to clarify the characteristics of t2d with ketosis in south - west rural parts of china . in summary , the study shows the characteristics of t2d with ketosis in adults of rural areas in south - west part of china . t2d with ketosis is a severe disorder of glucose and lipids metabolism and prone to acquire chronic complications . thus , there is an urgent and important need for the implementation of effective screening , health knowledge promotion , and control of diabetes .
aims . our group investigated albumin gene expression in human adipocytes , its regulation by inflammation and the possible contribution of adipose tissue to albumin circulating levels . methods . both inflamed and healthy subjects provided adipose tissue samples . rt - pcr , real - time pcr , and western blot analysis on homogenates of adipocytes and pre - adipocytes were performed . in sixty - three healthy subjects and fifty - four micro - inflamed end stage renal disease ( esrd ) patients circulating levels of albumin were measured by nephelometry ; all subjects were also evaluated for body composition , calculated from bioelectrical measurements and an thropometric data . results . a clear gene expression of albumin was showed in pre - adipocytes and , for the first time , in mature adipocytes . albumin gene expression resulted significantly higher in pre - adipocytes than in adipocytes . no significant difference in albumin gene expression was showed between healthy controls and inflamed patients . a significant negative correlation was observed between albumin levels and fat mass in both healthy subjects and inflamed esrd patients . conclusions . in the present study we found first time evidence that human adipocytes express albumin . our results also showed that systemic inflammation does not modulate albumin gene expression . the negative correlation between albumin and fat mass seems to exclude a significant contributing role of adipocyte in plasma albumin .	adipose tissue , once just believed a passive organ storing excess energy , is now considered a real endocrine organ , with an important role in the regulation of homeostatic systems . it secretes various proteins that can be classified into molecules acting on metabolic processes like glucose homeostasis and insulin sensitivity , and into inflammatory molecules like interleukin 6 ( il-6 ) , interleukin 1 , tumor necrosis factor- , c - reactive protein ( c - rp ) , leptin , and adiponectin . the adipose tissue may account for 2025% of systemic il-6 circulating levels and seems to be involved in the systemic inflammatory response associated with obesity , insulin resistance , and metabolic syndrome . on this basis , adipose tissue represents a new and fascinating multidisciplinary object of research . we previously found a clear expression of il-6 receptors and c - reactive protein in human adipocytes obtained from adipose tissue fragments of different districts ( subcutaneous and omental of noninflamed and inflamed patients ) , showing that the adipose tissue , and in particular the adipocyte , was not only responsible for generating an inflammatory state per se but it also represents a target , by itself , of systemic inflammation . we observed , in fact , that the adipocyte , stimulated by il-6 through its cell receptor produces c - rp , as it happens in the hepatocyte . adipose tissue is a mesodermallyderived organ that contains , in addition to adipocytes , a stromal population composed by different cell types , such as pre - adipocytes , stem cells , fibroblasts , and macrophages . mature adipocytes account for only 4060% of the whole cell population whatever the type of adipose tissue . pre - adipocytes are present throughout adult life in adipose tissue and can proliferate and differentiate into mature adipocytes , according to the energy balance ; they also share some features with macrophages , such as the capacity of phagocytosis in response to different stimuli . another important cell population of the adipose tissue is represented by mesenchymal stem cells ( mscs ) that are adherent , fibroblast - like , pluripotent , and nonhematopoietic progenitor cells . these cells retain the capacity to undergo into many mesenchymal cell types , including bone cells [ 8 , 9 ] , neuronal cells , adipose , and muscular tissue in vitro , and of note , into hepatocyte - like cells [ 1315 ] . thereby , both the pre - adipocyte , with a very restricted potential , and the multipotent mesenchymal cell represent adipocyte precursors . albumin , normally produced by the hepatocyte , is the most abundantly circulating protein in blood , where it plays an important role as binding protein and is involved in the regulation of colloid - osmotic homeostasis . it is also a negative acute phase protein and hypoalbuminemia is the result of the combined effects of inflammation , through inhibitory cytokine ( especially il-6 ) , and inadequate protein and caloric intake in patients with chronic inflammatory disease , such as chronic renal failure where it represents a cardiovascular risk factor . considered the well - known active role of adipocyte in c - rp production and the presumable link between adipocyte and hepatocyte , we aimed this study at investigating if mature adipocytes express the gene of albumin and at evaluating whether systemic inflammation might regulate this expression , as we previously found for c - rp . furthermore , we speculated whether the adipocyte might contribute to the total circulating levels of albumin , as it happens for il-6 . human subcutaneous adipose tissue samples were obtained from twelve subjects divided in two subgroups on the basis of a preliminary evaluation of c - rp circulating levels . according to pepys and hirschfield we assumed a cut - off level of 3 mg / l , in order to discriminate between inflamed and noninflamed patients . on the basis of c - rp values , we obtained a group of six healthy ( noninflamed ) subjects , without any clinical symptoms or sign of inflammation , who underwent minor surgery procedures and a group of six patients with chronic inflammatory disease who underwent elective surgical procedures : ( a ) four for cancer and ( b ) two patients operated for cholecystectomy . all enrolled subjects provided written , informed consent , and the ethics committee of our hospital approved the study . to avoid a possible confounding inflammatory effect exerted by obesity or overweight status , inflamed patients and noninflamed controls were bmi matched in both groups of subjects . the presence of other systemic diseases ( vasculitis , rheumatoid arthritis , osteoarthritis , and bowel or lung inflammatory disease ) was excluded in all subjects ( excepted malignancy ) ; in particular , before the definitive inclusion , the possible existence of any immunological disease , malignancy ( in the healthy group ) , and infectious disease was carefully investigated and excluded . blood samples were collected , before surgical procedures , from all studied subjects to obtain serum aliquots . adipose tissue biopsies were obtained during the surgical procedures in all 12 subjects and for each patient was obtained a sample of subcutaneous total white adipose tissue and a sample of omental adipose tissue . after removal , to get rid of tissue debris the bioptic material was washed twice with warm sterile 0.9% nacl solution ; then the sample was immediately frozen in liquid nitrogen and stored at 80c until rna extraction . c - rp circulating levels were determined by a high - sensitivity elisa assay ( bender medsystems , vienna , austria ) on serum aliquots of all subjects included in the study . the lower detection limit was 3 pg / ml , and the overall intra - assay coefficient of variation has been calculated to be 6.9% . the concentrations of il-6 in plasma samples were analyzed by elisa using a commercially available kit ( quantikine ; r&d systems , minneapolis , mn ) , as described elsewhere . the lower detection limit of il-6 assay was < 0.70 pg / ml , and the coefficient of variation of both inter- and intra - assay was < 5% . adipose tissue fragments obtained from the six noninflamed subjects and from the six inflamed patients were placed in 37c , sterile 0.9% nacl , 5.6 mm glucose , and 25 mm hepes buffer with ph adjusted to 7.4 and containing 50 u penicillin / ml plus 50 mg streptomycin / ml . the adipose tissue fragments were minced under sterile conditions and digested in krebs - ringer bicarbonate buffer supplemented with 5.6 mm glucose , 50 u penicillin / ml , 50 mg streptomycin / ml , and 17 mg type i collagenase/10 g adipose tissue ( worthington biochemical , lakewood , nj ) . digestion was for 75 min at 37c , with rotary agitation at 40 rpm . the isolated cells were filtered through a single layer of chiffon , and the isolated adipocytes were allowed to float for 5 min at 37c . the fluid under the floating adipocytes ( containing stromal fraction ) was transferred to a conical polypropylene 50-ml centrifuge tube . for the adipocytes , 1 ml of cell suspension of adipose cells was suspended in each tube containing specific adipocyte medium ( zen - bio , research triangle park , nc ) and incubated in the specific conditions until the cell lysis process . the stromal fraction was centrifuged at 800 g for 10 min . for the isolation of pre - adipocytes , we first centrifuged the cell pellet at 800 g for 10 minutes and then we washed three times with dulbecco 's modified eagle medium : nutrient mixture f-12 ( ham ) in combination 01 : 01 ( dmem : f12 , gibco - brl , carlsbad , california ) , which has been supplemented to give 15-mm nahco3 , penicillin 50-u : ml streptomycin and 50 mg : ml . after washing , the pellet was resuspended by trituration in dmem supplemented pre - adipocyte : f12 containing 10% fetal bovine serum ( fbs , gibco - brl , carlsbad , ca ) . the cells were then plated and incubated in a humidified incubator at 37c in an atmosphere of 5% co2 in air for 24 hours to allow attachment and proliferation of cells . after 24 hours , the medium was removed and replaced by specific pre - adipocyte medium ( zen - bio , inc . , research triangle park , nc ) and subculture . the genome sequences corresponding to albumin and -actin were obtained from genebank ( http://www.ncbi.nlm.nih.gov/ ) to identify specific primers . we analyzed the exon sequences to establish a pair of primers ( sense and antisense ) able to generate amplified fragments measuring between 150 and 700 bp in length , with annealing temperature between 55 and 62c . the sense and antisense primers were selected to include at least one intron to prevent genomic dna contamination during amplification . four g of total rna were subjected to cdna synthesis for 1 h at 37c using the ready to go you - primer first - strand beads kit ( amersham pharmacia biotech , piscataway , nj , code 27 - 9264 - 01 ) in a reaction mixture containing 0.5 g oligo - dt ( amersham pharmacia biotech cod . pcr amplification of cdna was performed in a reaction mixture containing 4 l of cdna sample and different primer sets ( 20 p / mol each ) . the amplification of albumin gene and human -actin gene , as an internal control , was achieved using 2 primer sets . after an initial denaturation at 94c for 5 min , pcr reactions were carried out using 30 cycles of 94c for 1 min , the temperature for annealing for 1 min , and 72c for 1 min in a perkin elmer cetus 480 thermal cycler ( perkin elmer ) . pcr products were separated by gel electrophoresis on a 2% agarose gel and stained with ethidium bromide . all signals were normalized to the mrna levels of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase and expressed as a ratio . real - time quantitative pcr analysis for albumin gene was performed on cell lysate ( both adipocytes and stromal fraction ) using abi prism 7500 ( applied biosystems , foster city , ca ) and the 5-exonuclease assay ( taqman technology ) . the cdna , synthesized as described above , was used for real - time pcr performed in 96-well optical reaction plates with cdna equivalent of 100 ng of rna in a volume of 25 l reaction containing 1x taqman universal master mix , optimized concentrations of fam - labeled probe , and specific forward and reverse primer for albumin gene from assay on demand ( applied biosystems ) . controls included rna subjected to rt - pcr without reverse transcriptase and pcr with water replacing cdna . the results were analyzed using a comparative method , and the values were normalized to the -actin expression and converted into fold change based on a doubling of pcr product in each pcr cycle according to the manufacturer 's guidelines , as previously described . homogenates of adipocytes and preadipocytes were prepared in radioimmune precipitation assay buffer ( 1x pbs , 1% np-40 , 0.5% sodium deoxycholate , 0.1% sds containing phenylmethylsulfonyl fluoride , aprotinin , sodium orthovanadate and protease inhibitor tablet ) ( completetm mini , boehringer - mannheim ) containing antipain dihydrochloride ( 50 mg / ml ) , bestatin ( 40 mg / ml ) , chymostatin ( 60 mg / ml ) , e-64 ( 10 mg / ml ) , leupeptin ( 0.5 mg / ml ) , pepstatin ( 0.7 mg / ml ) , phosphoramidon ( 300 mg / ml ) , pefabloc sc ( 1 mg / ml ) , edta disodium salt ( 0.5 mg / ml ) , and aprotinin ( 2 mg / ml ) ] . the cells were centrifuged at 14,000 g for 30 min at 4c and protein concentrations were determined with the bradford assay using bovine serum albumin as a standard . for immunoblotting , protein ( 100 g ) was subjected to 8% sds - polyacrylamide gel electrophoresis . proteins were transferred to polyvinylidene difluoride ( pvdf ) membranes at a constant voltage of 200 v for 16 h. the pvdf membranes were blocked for 1 h in 5% nonfat dried milk in tbs-0.1% tween buffer ( 25 mm tris - hcl , 0.2 mm nacl ; 0.1% tween 20 ( v / v ) ph 7.6 ) ( tbst ) . the membrane was washed 2x with tbst and then incubated overnight at 4c with the respective primary antibodies . albumin antibody was from cell signaling ( beverly , ma ) ; gadph antibodies were obtained from santa cruz biotechnology . after extensive washing of membrane with tbst buffer , anti - rabbit immunoglobulin conjugated with horseradish peroxidase was added at a 1 : 5000 dilution and incubated for 1 h at room temperature . an enhanced chemiluminescence kit ( amersham , nj ) was used to identify protein expression . in sixty - three healthy ( noninflamed ) subjects and fifty - four microinflamed ( c - rp : 5.24 2.26 mg / l ) esrd patients undergoing rdt we determined circulating albumin levels ( by nephelometry ) , body weight ( bw ) to the nearest 50 g ( by using a calibrated balance beam scale ) , body mass index ( bmi ) , calculated as the ratio body weight / height ( in kg / m ) , and body composition , assessed by conventional bioelectrical impedance analysis ( bia ) and by bioelectrical impedance vector analysis ( biva ) , as previously described . resistance ( r ) and reactance ( xc ) were measured by a single - frequency 50 khz bioelectrical impedance analyzer ( bia 101 rjl , akern bioresearch , firenze , italy ) according to the standard tetrapolar technique , by applying the software provided by the manufacturer , which incorporated validated predictive equations for total body water ( tbw ) , fat mass ( fm ) , fat free mass ( ffm ) , and extracellular water ( ecw ) [ 23 , 24 ] . the same investigators performed anthropometry and bia measurements . r and xc were normalized by the height of subjects ( r / h and xc / h ) and the resulting vectors were plotted on a graph reporting the gender - specific 50th , 75th , and 95th tolerance ellipses of similar vectors calculated from a reference healthy population . according to the rxc graph method , vectors falling within the reference gender - specific 75th tolerance ellipse indicated normal hydration ; short vectors ( below the lower pole of the 75th tolerance ellipse ) indicated overhydration and long vectors ( above the upper pole of the 75th tolerance ellipse ) indicated under - hydration . the length of the vector was calculated as \|z \| = [(r / h)2 + ( xc / h)2 ] and the phase angle of the vector as the arctan of xc / r . statistical analysis was performed by using unpaired t - test and anova ( followed by bonferroni post hoc test ) and linear regression analysis . results are expressed as means sd ; statistical significance was defined as p < 0.05 . demographic , anthropometric , and biochemical baseline data of all enrolled subjects are reported in table 2 . no significant difference was observed in sex , age , body weight , bmi , waist circumference , and waist - to - hip ratio between the two different groups . on the contrary , plasma c - rp and il-6 levels were much higher in patients with chronic inflammatory diseases than in healthy ( noninflamed ) subjects ( p < 0.01 ) . rt - pcr showed that the adipocytes extracted from all the districts of adipose tissue ( omental and subcutaneous ) of noninflamed controls and inflamed patients expressed the gene of albumin ( figure 1 ) ; the mrna for this marker was found in the adipocytes and pre - adipocytes of all fragments of adipose tissue . we did not find any statistically significant difference in albumin gene expression between inflamed and noninflamed patients in adipocytes drawn from both sc and om adipose tissue . as figure 2 shows , we only observed an higher omental adipocyte albumin than subcutaneous one in each group . we concluded that inflammation does not modulate albumin gene expression in the adipocyte , but probably further studies are required to confirm this result . figure 3 shows the different albumin gene expression , studied by real - time pcr , in adipocytes and pre - adipocytes obtained from either sc or om fragments of adipose tissue drawn from noninflamed subjects . pre - adipocyte albumin gene expression was higher then the adipocyte one in both om and sc fragments and this difference was statistically significant . we also observed that albumin gene expression in the pre - adipocyte extracted from fragments of om adipose tissue was significantly higher than in preadipocyte from sc fragments . finally , we investigated whether at the protein level adipocytes and pre - adipocyte were also able to express albumin in all the subjects . as shown in figures 4 and 5 , western blot analyses showed the presence of albumin in both adipocytes and pre - adipocytes suggesting that both adipocytes and pre - adipocytes can synthesize albumin in sc as well as in visceral adipose tissue , both in healthy noninflamed subjects and inflamed patients . we first investigated the relationship between plasma albumin levels and body fat mass ( fm ) in 63 healthy subjects who underwent bia excluding from statistical analysis over- and hypohydrated subjects ( vectors falling below the lower pole or vectors falling above the upper pole of the 75% tolerance ellipse ) evaluated by biva method . as shown in figure 6 , a significant negative correlation was observed between plasma albumin levels and fm ( r = 0.312 , p < 0.05 ( 2-tailed ) ) . then , we studied the same relationship in 54 microinflamed esrd patients undergoing regular dialysis therapy ( rdt ) . as shown in figure 7 , a significant negative correlation was observed between plasma albumin levels and fm ( r = 0.391 , p < 0.01 ( 2-tailed ) ) . albumin is the most abundant plasma protein produced by liver cells . to date , no data are present in the literature on albumin expression in mature adipocytes . in the present study , we found , for the first time , a clear albumin expression in human mature adipocytes . in addition , on the basis of our results , we can also reasonably affirm that differentiated adipocytes are probably able to synthesize albumin . albumin gene expression resulted significantly lower in the adipocytes than in the pre - adipocytes ; in particular , it was 42 e 12 times lower in om and sc adipocyte , respectively , as compared with the pre - adipocyte ( figure 3 ) . in this way , in fact , yoo and lee investigated the role of albumin in adipocyte differentiation , by using pre - adipocytes cell lines such as 3t3-l1 . this is a developmental process by which undifferentiated precursor cells differentiate into mature adipocytes with coordinated changes in cell morphology and gene expression . they found that albumin gene expression was significantly increased at later stages of adipocyte differentiation process and its suppression significantly inhibited lipid droplet formation . the author suggests that albumin could be necessary to stabilize lipid accumulation in mature adipocyte , probably through a direct interaction with fatty acids . however , it is important to underline that these experiments were performed on murine cell lines and their results can not correspond to human cell data . in a previous paper we found that not only c - rp gene expression was activated in adipocyte cells , but both il-6 and il-6 receptors gene expressions were found to be higher in inflamed patients than in controls , either in subcutaneous or intra - abdominal adipose tissue . at this regard , in the present study we hypothesized that adipocytes , similarly to hepatocytes , show a different albumin gene expression in inflamed and noninflamed patients , with a lower gene expression in inflamed ones . however , our results showed no significant difference in albumin gene expression between inflamed and noninflamed patients when analyzed by real - time pcr ( figure 2 ) . on the other hand , figure 2 shows also that albumin gene expression was significantly higher in intra - abdominal than in subcutaneous adipocytes ( figure 2 ) . albumin presence , as protein , together with gene expression in adipocytes raises the hypothesis that adipose tissue contribute to circulating albumin levels , as well as it happens for il-6 . to verify this hypothesis we evaluated the relationship between serum albumin and fat mass , supposing that higher fat mass corresponds to higher circulating albumin levels however , our results did not confirm this hypothesis , but we found a negative significant correlation between albumin and fat mass both in healthy noninflamed subjects and inflamed esrd patients , in contrast with what we expected ( figures 6 and 7 ) . in other words , the higher the fat mass the lower was serum albumin concentration was . we suppose that higher fat mass leads to higher production of different proinflammatory cytokines , mainly il-6 , that can downregulate albumin , production in hepatocyte via endocrine way , independently of systemic inflammation . we also suppose that il-6 produced by adipocyte could down - regulate , via an autocrine and/or paracrine signaling , albumin gene expression and production in the adipocyte itself . this mechanism could explain both the lack of modulation of albumin gene expression by systemic inflammation in the adipocyte and the negative correlation between fat mass and albumin levels either in noninflamed subjects or in inflamed esrd patients . on the contrary , this mechanism could not operate with inflammatory proteins , such as c - rp . despite the lack of albumin gene modulation by inflammation , in this study something really new was observed : the human adipocyte , once considered a simply depot cell , is now seen as a new and active cell , that in parallel with hepatocyte , is able to produce different proteins , such as c - rp and , as novelty , albumin . the more fascinating hypothesis states the existence of a continuum in adipose tissue cell population that goes from multipotent stem cell to more mature progenitor pools , passing through the pre - adipocyte . this hypothesis might explain why adipocyte and hepatocyte share the expression of some genes , such as albumin gene . as above mentioned , adipose - tissue - derived msc ( admsc ) displayed the capacity to differentiate into numerous cell types ( muscular , neuronal , bone , adipose cells ) and , interestingly , into hepatocyte - like cells [ 1315 ] . a study showed that admsc could be differentiated into functional hepatocyte - like cells by the treatment of cytokine mixtures in vitro , so to become a potential source to hepatocyte regeneration or liver cell transplantation . in another work , the authors showed that the undifferentiated nave admsc were also positive for albumin , g-6-p , and -1-antitrypsin ( aat ) , which are all known to be predominantly expressed in adult liver cells . however , we do suppose that other , more fascinating mechanisms , apart from sharing the same origin , may explain why , to date , adipocyte and hepatocyte produce a so important protein in our organism , like albumin . in conclusion , this preliminary study highlights for the first time a new adipocyte activity , among the others already known ; however further investigations are needed to confirm and explain our results .
south east asia pest thrips species , thrips parvispinus ( karny ) , is a serious pest on a number of agricultural and horticultural crops in a number of plant families . based on an integrated approach of morphology and dna barcoding , invasion of this serious pest is reported first time from india on papaya plantations . molecular data have corroborated with the morphological identification . haplotyping data suggested that the indonesia may be a probable source of invasion of this pest to india .	specimens were collected and stored in 70% ethanol at 80c . dna isolation and amplification of partial fragment of mtcoi gene , purification of amplified pcr products , and sequencing were performed as earlier protocol using non - destructive techniques ( buckman et al . voucher specimens have been retrieved after dna isolation and mounted in canada balsam onto glass slides and identified as t. parvispinus using the morphological keys ( mound 2005 ) . the voucher specimens have been submitted to national zoological collections , zoological survey of india , kolkata , india . eleven dna sequences generated in this study were aligned against 80 sequences of t. parvispinus from indonesia as retrieved from national centre for biotechnology information . further , the generated sequences were submitted to genbank database to acquire the accession numbers for t. parvispinus ( km485659km485667 ) and thrips orientalis ( km507077km507078 ) . t. orientalis is used as it is a member of the thrips orientalis group . evolutionary genetic divergences with kimura-2-parameter model and neighbor - joining ( nj ) phylogenetic tree were constructed in mega 6.0 with 1,000 bootstrap replications ( tamura et al . haplotyping was carried out in dnasp , and median joining networks were produced in network 4.1 ( bandelt et al . head broader than long , ocellar pair iii arising at the anterior margin of ocellar triangle ; postocular setae i and iv longer than iii ( fig . pronotum with two pairs of posteroangulars setae and two pairs of posteromarginal setae ( fig . metanotum reticulate medially and with faint internal reticules ; median setae long and placed behind the anterior margin ; campaniform sensilla absent ( fig . abdominal sternites iii vi with accessory setae , absent on ii and vii . figs . 13.t . parvispinus , female ( 1 ) head and pronotum ; ( 2 ) meso- and metanotum , ( 3 ) fore wing . t. parvispinus , female ( 1 ) head and pronotum ; ( 2 ) meso- and metanotum , ( 3 ) fore wing . thirteen females , two males , india : karnataka : bangalore , 10.ii.2014 , kamlajayanti ( reg . head broader than long , ocellar pair iii arising at the anterior margin of ocellar triangle ; postocular setae i and iv longer than iii ( fig . pronotum with two pairs of posteroangulars setae and two pairs of posteromarginal setae ( fig . metanotum reticulate medially and with faint internal reticules ; median setae long and placed behind the anterior margin ; campaniform sensilla absent ( fig . abdominal sternites iii vi with accessory setae , absent on ii and vii . figs . 13.t . parvispinus , female ( 1 ) head and pronotum ; ( 2 ) meso- and metanotum , ( 3 ) fore wing . t. parvispinus , female ( 1 ) head and pronotum ; ( 2 ) meso- and metanotum , ( 3 ) fore wing . thirteen females , two males , india : karnataka : bangalore , 10.ii.2014 , kamlajayanti ( reg . head broader than long , ocellar pair iii arising at the anterior margin of ocellar triangle ; postocular setae i and iv longer than iii ( fig . pronotum with two pairs of posteroangulars setae and two pairs of posteromarginal setae ( fig . metanotum reticulate medially and with faint internal reticules ; median setae long and placed behind the anterior margin ; campaniform sensilla absent ( fig . abdominal sternites iii vi with accessory setae , absent on ii and vii . figs . 13.t . parvispinus , female ( 1 ) head and pronotum ; ( 2 ) meso- and metanotum , ( 3 ) fore wing . t. parvispinus , female ( 1 ) head and pronotum ; ( 2 ) meso- and metanotum , ( 3 ) fore wing . thirteen females , two males , india : karnataka : bangalore , 10.ii.2014 , kamlajayanti ( reg . homology search using blast search option resulted in 99100% similarity to t. parvispinus sequences from indonesia . we analyzed 91 partial mtcoi sequences of t. parvispinus and t. orientalis in this study . out of which 11 sequences were generated in this study and rest of the 80 sequences were retrieved from national centre for biotechnology . complete dataset after trimming have 604 nucleotides , which shows 89 variable sites with 87 parsimony informative sites . however , the dataset of 89 sequence of t. parvispinus yielded only 14 variable sites out of which 10 were parsimony informative . analysis of 89 sequences of t. parvispinus yielded four haplotypes ( hap_1 to hap_4 ) ( fig . the data show that there is no host - plant or geographical locality specific haplotyping . the total number of segregating sites between four derived haplotypes varies from 1 to 13 ( table 1 ) . further , out of 14 segregating sites , 11 were detected as synonymous changes and 3 as nonsynonymous changes corresponding to nine transitions and five transversions . the analysis of nj tree yielded two major clades with high bootstrap support ; clade i includes 89 sequences of t. parvispinus from indonesia and india , while clade ii is represented by two sequences of t. orientalis ( fig . nj tree provided here is only to segregate two species based on the reciprocal monophyly criteria and not to interpret phylogeny of genus thrips . fig . table 1.segregating nucleotide sites in the t. parvispinus mtcoi nucleotide sites as depicted in four haplotypeshaplotypes166672104123150165179279360364495537570hap_1 ( indonesia ) , n = 63catattagatttathap_2 ( indonesia , india ) , n = 18 ......... c .... hap_3 ( indonesia ) , n = 7tg .. c.g.gccctahap_4 ( india ) , n = 1 .. gg.g.t.c .... transversions are shown in bold . f. schultzei is used as an outgroup . segregating nucleotide sites in the t. parvispinus mtcoi nucleotide sites as depicted in four haplotypes transversions both morphological and molecular evidences verify that the specimens collected on papaya represents t. parvispinus . the presence of this pest species on an economically important crop plant like papaya in india raise serious issues and is a concern for quarantine authorities . occurrence of t. parvispinus in other parts of india needs systematic monitoring as it is likely to acquire pest status in future . molecular evidence for shared haplotype ( h_2 ) between indonesia and india indicated that there is a flow of genetic material , and indonesia may be a probable source of invasion of this species to india . however , molecular data on this species from other countries may be helpful to trace exact route of invasion .
background : the aim of this study was to investigate the effect of hemiscorpius lepturus venom on leukocytes and the leukocyte subgroups in peripheral blood of rat.methods:in this experimental study , sixty n - mari rats were divided into three groups of 20 rats . then the rats in each group were divided into four subgroups based on the blood sampling time that was 2 , 6 , 24 and 48 hours after the venom injection , respectively . the control group did not receive anything , however , the first and the second experimental groups received 0.1 and 0.01mg / kg of venom , subcutaneously . in accordance with a designated four sampling times , the blood sampling was carried out in three groups . after rbc lysis , the leukocytes and leukocyte sub - populations were determined and counted using appropriate hematological standard methods.results:the leukocyte and the neutrophil count at two ( p<0.05 ) , six ( p<0.01 ) and 24 ( p<0.05 ) hours after the venom injection showed a significant decline compared with the control group , this decrease was significant at the dose of 0.1 mg / kg until 48 hours after the venom injection ( p<0.05 ) . the lymphocyte count showed a significant decline throughout the all hours of the experiment , compared with the control group ( p<0.05).conclusion : leukocytes are probably affected by the cytotoxicity effect of the h. lepturus venom in a dose - dependent manner . this could be a wakeup call for the medical staff to perform quick and accurate treatment in the least time possible .	scorpion envenomation is one of the main problems in the public health system in many countries in the world . this involves 2.3 billion inhabitants in the areas with the scorpion sting threat ( chippaux et al . 2008 ) . in 2008 , the annual incidence of scorpion stings was 1.200.000 leading to 3250 deaths ( chip - paux et al . this number demonstrated a relative growth in a report released in 2012 and exceeded than annually 1,500,000 scorpion stings , however , the mortality rate due to the scorpion stings showed a significant decrease and fell into 2600 deaths per year ( jean - philippe 2012 ) . the highest number of scorpion sting in the world has been allocated to iran after mexico ( osnaya - romero et al . the annually 42,500 scorpion stings and 20 following deaths have been reported from 2001 to 2009 , in iran ( celis et al . 2007 ) . in the middle east , among the 52 known species of scorpions , the most dangerous scorpions are reported in iran ( celis et al . 2007 ) . the scorpion sting has been reported from all of the provinces of iran , however , the most common incidence rates have been detected in khuzestan , with incidences of 541 per 100000 individuals ( dehghani et al . hemiscorpius lepturus belongs to the hemiscorpiidae family , and is the most medically important and a dangerous scorpion in khuzestan , iran and the world ( shahbazzadeh et al . hemiscorpius lepturus has been responsible for 15 % of the scorpion sting bite cases , however , it is the leading cause of 89 % of deaths followed by the scorpion sting ( pipelzadeh et al . the lethality arising from this scorpion is approximately 60 times higher than the average for the remaining venomous scorpion stings in the region ( pipelzadeh et al . the venom of h. lepturus leads to acute renal failure , thrombocytopenia and microangiopathic hemolytic anemia , known as the nephrotoxic , hepatotoxic and hemolytic complications of the scorpion venom ( valavi et al . the local signs vary from erythema to necrosis , while the patient feels no pain . on the other hand , the nephrotoxicity is the most important systemic complication that if left untreated could result in severe renal , cardiac and pulmonary failure ( pipelzadeh et al . . the venom may induce severe pathological damages in target organs such as skin , blood cells , central nervous system ( cns ) , and cardiovascular system ( seyedian et al . it leads to an increase in liver enzymes aspartate aminotransferase ( ast ) , alanine aminotransferase ( alt ) and alkaline phosphatase ( alp ) , indicating the severe hepatic damage ( pipelzadeh et al . showed that the h. lepturus causes more rbc lysis and ldh increase , comparing with the complications of envenomation resulting from the stings of two other species of scorpions , mesobuthus eupeus and androctonus crassicauda ( khodadadi et al . , it was described that 90 % of patients whom were admitted in hospitals due to the general condition worsening , were stung by h. lepturus ( mir dehghan et al . the immune system cells are distributed throughout the body , from the outer most points to the deepest organs and tissues , such as blood , bone marrow , thymus and spleen . despite this diversity , these cells apply a comprehen - sive supervision on different organs and tissues through the regular circulation from blood to lymph , from inside of the vessels to the outside and from the interior of the organs to the blood , resulting in protection of the body against the pathological factors ( vodjgani 2012 ) . the results of an experimental study aiming the investigation of the effect of envenomation by h. lepturus on the hematological indices three days after injection of the venom suggested that the leukocyte number has been increased over the normal range , however , there was not any significant difference with the control group ( dehghani et al . an increase in the peripheral leukocyte count had been demonstrated during the investigation of the blood among h. lepturus scorpion sting victims ( chitnis et al . 1993 ) . while comparative studies to investigate the effects of h. lepturus venom on hematologic parameters and vital organs of the body have been done , but so far , any experimental research on the effect of the scorpion venom on the immune system cells and its various sub - groups in the early hours after the sting , and further - more determination and investigation of the period and the intensity of the envenomation have not carried out yet . therefore , the aim of the present study was to investigate the effect of h. lepturus envenomation on blood leukocytes and their subgroups in the early hours after the sting . the results of this study can help physicians , health officials and the medical staff to fast and accurate treatment of the victims of h. lepturuss sting and prevention of complications of the scorpion venom on important factors of the immune system . sixty male rats from n - mari species ( weight range of 300350 grams ) purchased from pasteur institute of iran ( tehran ) , were used during the study . the animals were kept in standard cages in animal house at the school of pharmacy of ahvaz jundishapur university of medical sciences . rats were housed in temperature - controlled rooms ( 2225 c ) with constant relative humidity ( 4070% ) and 12h/12h light / dark cycle before doing experimental protocols . the study was performed in accordance with the principles of laboratory care established by the ethics committee of ahvaz jundishapur university of medical sciences , ahvaz , iran . the scorpion electroshocked venom was provided as a lyophylized powder from pasteur institute of iran , ( venom and therapeutic biomolecule lab , biotechnology res . the concentration of crude venom protein was determined by using bradford method ( bradford , 1976 ) . the injection volume was 0.1 ml . the animals were randomly divided into three groups of 20 rats in each . the control group did not receive any thing ; however , the first and the second groups received h. lepturus venom at the concentrations of 0.1 and 0.01 mg / kg / bw subcutaneously . thereafter , the animals of each group ( n= 20 ) were divided into four subgroups ( n= 5 ) , with respect of the four blood sampling time , that were two , six , 24 and 48 hours after the venom injection , the animals were then kept in separate cages . the animals of each subgroup were anesthetized with ketamine and xylazine ( alfasan , holland ) . the blood samples were obtained from the animal s heart amounted to 0.52 ml by syringe . soon after the sampling , the blood was maintained in glasses containing the anticoagulant , edta ( ethylene - diamine - tetra - acetic acid ) , and the leukocytes were counted using the diluent solution , marcanu ( rbc lysis buffer ) and the neobar slide ( hemocytometer ) by using the light microscope ( olympus , 3h - z - japan ) , in order to count and determine the leukocyte subgroups ( including neutrophils , lymphocytes , monocytes and eosinophils ) the appropriate peripheral blood smears were prepared on the microscope slides and then fixed by means of the water - free methanol . then giemsa staining ( merck - germany ) was carried out by the diluted stain with the rate of 1/10 . and finally , the differential counting was performed using a 100x lens microscope ( mahbod 2008 , mansouri et al . 13 ( version 13 , spss inc , chicago , il ) and the statistical tests of anova and lsd . 15 . the comparison of the meansd of white blood cells ( 10 per ml ) in peripheral blood of rat between the groups receiving venom with the concentration of 0.01 and 0.1 mg / kg and the control group at different times significant difference ( p < 0.05 ) between the experimental and control groups * * significant difference ( p < 0.01 ) between the experimental and control groups the comparison of the meansd of neutrophils ( 10 per ml ) in peripheral blood of rat between the groups receiving venom with the concentration of 0.01 and 0.1 mg / kg and the control group at different times * significant difference ( p < 0.05 ) between the experimental and control groups * * significant difference ( p < 0.01 ) between the experimental and control groups the comparison of the meansd of lymphocytes ( 10 per ml ) in peripheral blood of rat between the groups receiving venom with the concentration of 0.01 and 0.1 mg / kg , and the control group at different times * significant difference ( p < 0.05 ) between the experimental and control groups significant difference ( p < 0.01 ) between the experimental and control groups the comparison of the meansd of monocytes ( 10 per ml ) in peripheral blood of rat between the groups receiving venom with the concentration of 0.01 and 0.1 mg / kg and the control group at different times the comparison of the meansd of eosino - phil ( 10 per ml ) in peripheral blood of rat between the groups receiving venom with the concentration of 0.01 and 0.1 mg / kg and the control group at different times sixty male rats from n - mari species ( weight range of 300350 grams ) purchased from pasteur institute of iran ( tehran ) , were used during the study . the animals were kept in standard cages in animal house at the school of pharmacy of ahvaz jundishapur university of medical sciences . rats were housed in temperature - controlled rooms ( 2225 c ) with constant relative humidity ( 4070% ) and 12h/12h light / dark cycle before doing experimental protocols . the study was performed in accordance with the principles of laboratory care established by the ethics committee of ahvaz jundishapur university of medical sciences , ahvaz , iran . the scorpion electroshocked venom was provided as a lyophylized powder from pasteur institute of iran , ( venom and therapeutic biomolecule lab , biotechnology res . the concentration of crude venom protein was determined by using bradford method ( bradford , 1976 ) . the control group did not receive any thing ; however , the first and the second groups received h. lepturus venom at the concentrations of 0.1 and 0.01 mg / kg / bw subcutaneously . thereafter , the animals of each group ( n= 20 ) were divided into four subgroups ( n= 5 ) , with respect of the four blood sampling time , that were two , six , 24 and 48 hours after the venom injection , the animals were then kept in separate cages . the animals of each subgroup were anesthetized with ketamine and xylazine ( alfasan , holland ) . the blood samples were obtained from the animal s heart amounted to 0.52 ml by syringe . soon after the sampling , the blood was maintained in glasses containing the anticoagulant , edta ( ethylene - diamine - tetra - acetic acid ) , and the leukocytes were counted using the diluent solution , marcanu ( rbc lysis buffer ) and the neobar slide ( hemocytometer ) by using the light microscope ( olympus , 3h - z - japan ) , in order to count and determine the leukocyte subgroups ( including neutrophils , lymphocytes , monocytes and eosinophils ) the appropriate peripheral blood smears were prepared on the microscope slides and then fixed by means of the water - free methanol . then giemsa staining ( merck - germany ) was carried out by the diluted stain with the rate of 1/10 . and finally , the differential counting was performed using a 100x lens microscope ( mahbod 2008 , mansouri et al . 13 ( version 13 , spss inc , chicago , il ) and the statistical tests of anova and lsd . 15 . the comparison of the meansd of white blood cells ( 10 per ml ) in peripheral blood of rat between the groups receiving venom with the concentration of 0.01 and 0.1 mg / kg and the control group at different times significant difference ( p < 0.05 ) between the experimental and control groups * * significant difference ( p < 0.01 ) between the experimental and control groups the comparison of the meansd of neutrophils ( 10 per ml ) in peripheral blood of rat between the groups receiving venom with the concentration of 0.01 and 0.1 mg / kg and the control group at different times * significant difference ( p < 0.05 ) between the experimental and control groups * * significant difference ( p < 0.01 ) between the experimental and control groups the comparison of the meansd of lymphocytes ( 10 per ml ) in peripheral blood of rat between the groups receiving venom with the concentration of 0.01 and 0.1 mg / kg , and the control group at different times * significant difference ( p < 0.05 ) between the experimental and control groups significant difference ( p < 0.01 ) between the experimental and control groups the comparison of the meansd of monocytes ( 10 per ml ) in peripheral blood of rat between the groups receiving venom with the concentration of 0.01 and 0.1 mg / kg and the control group at different times the comparison of the meansd of eosino - phil ( 10 per ml ) in peripheral blood of rat between the groups receiving venom with the concentration of 0.01 and 0.1 mg / kg and the control group at different times the scorpion venom has led to a significant reduction of leukocytes in the venom - receiving group with the dose of 0.01 mg / kg at two and 24 hours after the injection , compared with the control group ( p < 0.05 ) . this decrease has been maximum of six hours after the injection ( p < 0.01 ) . after 24 hours from the injection , the effect of the venom has subsided ; and after 48 h there was no any significant difference with the control group ( p > 0.05 ) . the toxicity effect of the venom is intensified with the increase of the injected venom concentration , so that in spite of a decrease in venom effect in the group receiving 0.01 mg / kg after 48 hours , the decrease in toxicity effect is still significant in the group receiving 0.1 mg / kg compared with the control group ( p < 0.01 ) ( fig . the neutrophil mean count showed a significant decline in the venom receiving group with the dose of 0.01 mg / kg at two , 24 and 48 hours ( p < 0.05 ) , and six hours after the injection , compared with the control group ( fig . the mean of the peripheral blood lymphocyte count in the venom receiving group of 0.01 mg / kg at two , six and 24 hours ( p < 0.05 ) , and 48 hours ( p < 0.01 ) after the injection demonstrated a significant decline compared with the control group , respectively . the comparison of the mean and standard deviation of the eosinophil and monocytes of the peripheral blood in the case and control groups showed no significant differences ( p > 0.05 ) ( figs . 4 , 5 ) . the scorpion venom has led to a significant reduction of leukocytes in the venom - receiving group with the dose of 0.01 mg / kg at two and 24 hours after the injection , compared with the control group ( p < 0.05 ) . this decrease has been maximum of six hours after the injection ( p < 0.01 ) . after 24 hours from the injection , the effect of the venom has subsided ; and after 48 h there was no any significant difference with the control group ( p > 0.05 ) . the toxicity effect of the venom is intensified with the increase of the injected venom concentration , so that in spite of a decrease in venom effect in the group receiving 0.01 mg / kg after 48 hours , the decrease in toxicity effect is still significant in the group receiving 0.1 mg / kg compared with the control group ( p < 0.01 ) ( fig . the neutrophil mean count showed a significant decline in the venom receiving group with the dose of 0.01 mg / kg at two , 24 and 48 hours ( p < 0.05 ) , and six hours after the injection , compared with the control group ( fig . the mean of the peripheral blood lymphocyte count in the venom receiving group of 0.01 mg / kg at two , six and 24 hours ( p < 0.05 ) , and 48 hours ( p < 0.01 ) after the injection demonstrated a significant decline compared with the control group , respectively . the comparison of the mean and standard deviation of the eosinophil and monocytes of the peripheral blood in the case and control groups showed no significant differences ( p > 0.05 ) ( figs . 4 , 5 ) . the present study was aimed to investigate the effect of h. lepturus venom on leukocytes and their subgroups in peripheral blood of rats . these cells apply a comprehensive supervision on different organs and tissues , through the regular circulation of blood to lymph , from inside of the vessels to the outside and from the interior of the tissues to the blood , resulting in a protection of the body against the pathological factors . in general , leukocytes consist of various groups of cells including lymphocytes , monocytes and granulocytes ( vodjgani 2012 ) . the clinical syndrome induced by h. lepturus sting is different from the stings by the other scorpions existing in iran and the world , and exhibits more severe manifestations . lack of local pain or being a mild pain after the sting , cutaneous manifestations such as erythema , swelling and necrosis at the sting site , the red blood cell lysis , and nephrotoxicity , including hemoglobinuria , proteinuria and hematuria are some manifestations among scorpion sting victims ( radmansh 1990 , radmanesh 1998 , pipelzadeh et al . the results of the present study demonstrated those leukocytes are affected by the venom after two hours , exhibiting sensitivity , decrease in leukocytes continues for six hours after the venom injection . if leukocytes were evaluated after 12 hours , it was possible to notice a decreasing trend , due to prolongation of the presence of the venom into the body . probably these variations in leukocytes are the results of cytotoxic effects of h. lepturus venom , which leads to white blood cell lysis and destruction ( shayesteh et al . there is no any considerable experimental or clinical study in this field , and the decrease in leukocytes in the early hours is one of the new findings of this research . lack of the related reports might be due to the difference in evaluation time of the hematological indices in various studies ( dehghani et al 2005 , dehghani et al . 2012 ) , it has been the result of the use of the anti - venom in human researches , as well ( chitnis et al . the leukocyte count trend increased after 24 hours of venom injection , and there was not any significant difference in the venom - receiving group of 0.01 mg / kg with the control group . various studies have reported leukocytosis as well as the major clinical signs of the envenomation by h. lepturus , after other symptom such as hemoglobinuria , hematuria and proteinuria ( chitnis et al . therefore , the reconstruction of leukocytes observed in this study is in agreement with the previous findings ( dehghani et al . 2012 ) . however , the more venom concentration , the more decline in leukocyte count , the slower wbc reconstruction , so that the leukocyte count has not reached to the normal range after 48 hours of envenomation with 0.1 mg / kg . according to the present study , after two hours , the percent of the blood neutrophils has been changed from the normal range of 61 % to 35.2 % . this reduction has been more severe after six hours and has fallen down to 25.5 % . neutrophils make up the most abundant population of white blood cells , and mediate the primary stages of inflammatory response . they are the most effective phagocytes in peripheral blood and have a major role in defense against the extracellular factors ( abbas et al . these mature cells migrate to the inflammation site just four hours after the antigen entrance , and have the capability to invade the antigen . phagocytosis of the particles and waste products in neutrophils is associated with a series of biochemical events and morphological changes in the cell ( vodjgani 2012 ) . the majority of the neutrophils at the inflammation site will be wiped out by other cells , such as macrophages , after the phagocytosis of the invader . 2011 aiming to investigate the effect of h. lepturus on the serum levels of cytokines il-1 , il-6 , il-8 and tnf- , demonstrated a direct relation between the worsening of the patient s general condition and the above - mentioned cytokines . il-8 is a chemotactic protein that is known as nap-1 or the attractive and activator of neutrophils ( vodjgani 2012 ) . the increase of this cytokine leads to fever and hyperthermia , which is probably due to neutrophil aggregation , pathogen killing and the death of the two kinds of cells ( taraz 2008 ) . tnf- enhances the production of some particular serum proteins such as amyloid a , through affecting hepatocytes . this cytokine suppresses stem cell division , which may lead to neutropenia ( vodjgani 2012 ) . therefore , the severe decrease in neutrophils in the group receiving high doses of h. lepturus venom will be reasonable . in the following 24 hours , the neutrophil count especially in the venom - receiving group of 0.01 mg / kg has approached the normal range . regarding the neutrophilia , the neutrophil compensation could be explained . through the neutrophilia the conversion of marginal neutrophils into circulating neutrophils could be noticed . these findings are consistent with the results of the research carried out by ghafourian and mohebby on patients with scorpion referred to the hospital bite ( ghafourian and mohebbi 2008 ) . in the present study , lymphocytes have decreased during the first hours after the venom injection , and this reduction was significant even after 48 hours . one of the fractions extracted from the venom of h. lepturus may reduce the lymphocyte count ( bigdeli et al . lymphocytes make up around 20 to 40 % of the leukocytes , and 99 % of the cells in the lymph , respectively . the process of recognition and processing of antigen by lymphocytes as well as the clonal expansion requires the time , and the precise determination of the mechanism of the effect of the venom of h. lepturus on lymphocytes can not be explained during the first 48 hours , perhaps due to immunological reasons . in the present study , the blood lymphocyte decrease was dose - dependent , which could probably be the result of the direct effect of the venom on the lymphocytes , which leads to disruption and lysis of these cells . in fact , it could be concluded that the venom of h. lepturus has a lymphotoxic effect ( ghafourian and mohebbi 2008 ) . in the present research , the peripheral blood eosinophil count in the control group showed no significant difference with the experimental groups . eosino - phil consists of 2 % of leukocytes found in the normal situation in tissues , especially the epithelium of the respiratory tract , gastrointestinal tract and genitourinary tract . these cells have a weak phagocytic ability , and increase mainly in type-1 hypersensitivity responses and parasitic infections , as well . the venom of the scorpion h. lepturus effect on the leukocytes in the early hours , as well as increasing the concentration of the toxin , its destructive power is increased . in other word however , the essential role of these factors is to intensify the inflammation , but recently the natural corticosteroid hormones or similar synthetic substances are used to alleviate the inflammatory reactions against the allograft transplantation and immune system suppression . therefore , the extraction of the useful fractions of h. lepturus venom for natural induction of corticosteroids and decreasing leukocytes may be useful in the treatment of some types of leukemia and the graft surgeries , as well . however , the severe and dose - dependent reduction of the immune cells in the first hours after the injection of h. lepturus venom could be an alarm for health officials and medical staff to perform quick and accurate treatment in the least possible time , and to prevent of complications of the scorpion venom is the body s vital organs .
plasma cell neoplasms result from monoclonal proliferation of plasma cells . solitary extramedullary plasmacytomas ( semps ) are rare and constitute 5% of all plasma cell disorders . semps most commonly involve upper aerodigestive tract . isolated spinal epidural space involvement by semps is extremely rare and to best of our knowledge only 7 such cases have been reported previously in available english literature . we hereby present a rare case of thoracic epidural semp in a 32-year - old female who presented with thoracic compressive myelopathy and discuss the pertinent literature .	solitary plasmacytomas ( sp ) result from monoclonal plasma cell proliferation but lack bone marrow involvement and m protein in serum / urine , seen in multiple myeloma . sp has been classified by world health organisation into solitary osseous plasmacytomas ( sop ) and solitary extramedullary plasmacytomas ( semp ) . semps are rare and constitute 5% of all plasma cell disorders and have a strong predilection for the upper aerodigestive tract . we hereby describe one such rare case of thoracic epidural semp manifesting as dorsal compressive myelopathy . a 32-year - old female presented to us with a back pain for 2 months and progressive spastic weakness of bilateral lower limbs ( b / l lls ) for past 8 days . clinical examination revealed spastic weakness ( power : 1/5 ; mrc uk ) of b / l lls , exaggerated b / l knee and ankle jerks and complete sensory loss at and below l1 level . spine examination revealed no deformity or tenderness . with a clinical diagnosis of thoracic compressive mri revealed a dorsally located epidural lesion at the level of t7-t8 vertebral bodies , which was compressing and pushing the spinal cord anterolaterally [ figure 1a and b ] . the lesion was isointense on t1-weighted , hypointense on t2-weighted images and enhanced homogenously and extended into the neural foramen . both meningiomas and neurofibromas are isointense to hyper intense and not hypo intense on t2-weighted images . magnetic resonance imaging ( mri ) showing a epidural lesion at the level of t7-t8 vertebrae ( a , sagittal t2-weighted ) , pushing the cord anterolaterally to right side and extending into the neural foramen ( b , axial t2-weighted ) . postoperative mri ( sagittal t1-weighted , parasgittal t2-weighted and axial t1-weighted images ) revealed complete excision of the tumor with opening of the subarachnoid space ( c - e ) at the level of tumor . the spinal cord can be seen to have attained normal shape and position ( e ) the patient underwent t7 and t8 laminectomy and complete excision of tumor , which was reddish , soft , moderately vascular and located in epidural space with extension into the left neural foramen . as there was no bony involvement and only two level laminectomy was done , no vertebral stabilization was needed . histopathological examination revealed diffuse and dense infiltration by mature and immature plasma cells which were immunohistochemically positive for cd138 , with occasional bi - nucleated plasma cell . based on overall histomorphological and immunohistochemical findings , the diagnosis of plasmacytoma was made [ figure 2 ] . ( a ) microphotograph showing a tumor composed of diffuse infiltration by both mature and immature plasma cells ( h and e , 20 ) ( b ) plasma cells are highlighted by immunohistochemical staining for cd138 ( immunohistochemistry , 40 ) patient was then evaluated to find a systemic evidence of disease . bone marrow examination revealed < 5% plasma cells and serum electrophoresis and urine examination were negative for m protein . patient received adjuvant radiotherapy ( rt ) ( 40 gy in 20 fractions ) to operative field . 6 months after surgery patient has started walking ( power in bilateral lower limbs : 4/5 mrc uk ) and has regained bladder functions . solitary extramedullary plasmacytomas most commonly involve upper aerodigestive ( 8090% of cases ) . in 1020% cases other body organs , including skin , testis , ovaries , liver , lungs , spleen etc . however , occurrence of semps as isolated masses in spinal epidural space is very rare . thorough search of the literature revealed only seven such cases reported previously in available english literature [ table 1 ] . cases of epidural semps in order to make a diagnosis of semp , following criteria must be fulfilled : biopsy from lesion showing monoclonal plasma cellsbone marrow plasma cell < 5%absence of osteolytic bone lesions or involvement of other body tissuesabsence of hypercalcemia and renal failureabsent or low serum m - protein concentration . biopsy from lesion showing monoclonal plasma cells bone marrow plasma cell < 5% absence of osteolytic bone lesions or involvement of other body tissues absence of hypercalcemia and renal failure absent or low serum m - protein concentration . radiologically , the epidural mass can be a diagnostic dilemma . in an endemic country , tuberculosis is the first consideration in the differential diagnosis , and we also considered it as first differential . treating these patients with only antitubercular drugs is not only a futile exercise , it also has adverse consequences in terms of delaying the appropriate treatment for plasmacytoma . it is therefore important to be aware of this rare entity as a differential diagnosis of epidural masses especially when a t2-weighted hypo intense lesion is restricted to epidural space without bony involvement and without associated paravertebral collection . among the 7 cases in literature and the present case [ table 1 ] , both males and females are equally represented with an average age of 51 years ( age range : 4085 years ) . four patients had impairment of bladder and bowel functions . surgical resection followed by adjuvant therapy ( chemotherapy [ ct ] , rt or a combination of ct and rt ) , surgical resection alone , a combination of plasmapharesis , ct and rt , ct and rt and ct alone were different treatment regimens used in these patients [ table 1 ] . among the six patients with neurological deficits ( paraplegia / quadriplegia ) three patients undergoing surgical resection showed improvement in neurological deficits while other three who received ct or combined ct / rt did not show any neurological improvement [ table 1 ] . according to the guidelines by the british committee for standards in hematology , rt is the treatment of choice for sops and semps . the role of surgery is in providing a tissue diagnosis and in cases of spinal epidural semps , surgical resection relieves the compression on spinal cord thereby leading to neurological improvement . epidural semps are very rare and require a high index of suspicion for diagnosis and appropriate management as both clinical manifestations and radiological features can be quite similar to other more common pathologies in this region . though rt is the treatment of choice for sp , surgical resection helps in providing tissue diagnosis and relieving spinal cord compression .
editor s note : there is not just one type of epilepsy . while some forms of the disease are characterized by convulsive seizures , others involve seizures that are barely noticeable . seizures can occur for many reasons : they can be caused by genetic mutations , injury , or infection early in life . in addition , events in daily life , such as stress , or normal variations in hormones , such as estrogen and testosterone , can influence brain activity and therefore influence seizures . by considering the powerful interactions between the brain and the endocrine system , this influence of hormones on seizures can be understood and new treatment options can be considered .	the relationship between the gonads ( ovaries and testes ) and the brain is complicated . it is not surprising that we still do not completely understand all of the effects of hormones on the brain or on seizures . reproductive hormones like estrogen , progesterone , and testosterone have profound effects on the brains of both men and women , even in parts of the brain that are not considered to be critical to reproduction . moreover , these effects occur throughout life , not only in adulthood . during early stages of development , researchers have shown that in laboratory animals , estrogen levels in the fetus are critical to the gender of the fetus.7 after puberty , levels of circulating estrogen and testosterone also affect the brain ; the interplay between the early , organizational effects and the postpubertal , activating effects of hormones mediates the net effect of hormones in the adult brain.8,9 besides being synthesized in the gonads , estrogen , progesterone , and testosterone can be synthesized elsewhere , including in the brain itself . such synthesis can potentially occur anywhere in the brain because it occurs in astrocytes , cells that are located in all areas of the brain . astrocytes have historically been considered supportive cells , responsible for housekeeping in the brain ( cleaning up debris , removing damaged cells or infection ) . we now know that astrocytes have many other functions , including the synthesis of hormones , because enzymes in astrocytes metabolize cholesterol , a component of the outer membranes of all cells , into hormones . we do know that both estrogen and testosterone have many effects on the brain , influencing neurotransmitters ( which allow neurons to communicate ) and neuronal activity.10,11 hormones also have metabolites that have effects on brain activity . for example , allopregnanolone , a metabolite of progesterone , inhibits neuronal activity in many areas of the brain . as a result , it has been suggested that progesterone would be help stop seizures.12 because reproductive hormones affect brain activity , the changes in hormones at puberty , pregnancy , or during the menstrual cycle are important to consider when treating women with epilepsy . treatment is complicated , however , because hormones like estrogen and progesterone do not affect all patients in the same way . for example , some women experience changes in the severity and frequency of seizures in relation to estrogen or progesterone treatment , but others do not . similarly , some women have seizures that worsen during part of their menstrual cycle , but some do not . furthermore , some effects of hormones on seizures that have been demonstrated in laboratory experiments are not reproducible . for example , sometimes estrogen appears to promote seizures , but it does nt always.12 besides confusion , this has led to frustration , because clinicians are unsure how to treat patients whose seizures have suddenly worsened , possibly because of changes in reproductive hormone levels . nevertheless , our understanding of effects of estrogen and progesterone on the brain has led to hypotheses that explain why seizures can increase at certain times during the menstrual cycle . as mentioned above , catamenial epilepsy refers to seizures that worsen at particular times during the menstrual cycle . this syndrome has been recognized for more than 100 years , but it has been difficult to study.13 some investigators have even questioned whether catamenial epilepsy exists.14 many clinicians do not find robust sex differences in their patients,1517 and they therefore conclude that syndromes like catamenial epilepsy are not likely to be significant.13 however , clinical research provides strong evidence that catamenial epilepsy is common . andrew herzog , a leader in this field , estimates that catamenial epilepsy occurs in one - third of women with epilepsy.2 a recent epidemiological study in korea estimated that catamenial epilepsy occurs in almost 50 percent of women with epilepsy.18 the accuracy of these estimations is hard to judge , however , because seizures that are unnoticed will be missed , leading to an underestimation of the number of women with catamenial epilepsy . nevertheless , when the condition has been documented , symptoms are often the worst during the days just before the start of menstruation , approximately day 28 of the menstrual cycle , and the days just after the onset of menstruation the perimenstrual period . symptoms also worsen during the periovulatory phase , approximately day 14 , when ovulation occurs ( see diagram 1 , page 9 ) . the rise in circulating estrogen ( serum estrogen ) during the menstrual cycle affects the brain in many ways , and includes effects on neurons and non - neuronal cells such as astrocytes . in neurons , cell structure spines are important because the spines receive excitatory input from other neurons and help neurons communicate with one another . many scientists believe that a major effect of estrogen in the brain is increased neuronal activity because estrogen increases dendritic spines . circulating progesterone has many effects , because progesterone itself has actions on neurons , and its metabolite allopregnanolone does also . allopregnanolone binds to receptors that are responsible for effects of gamma - aminobutyric acid ( gaba ) , the major inhibitory neurotransmitter in the brain . therefore , when progesterone rises during the menstrual cycle , it is thought that neuronal activity will decrease in many parts of the brain . most of our current understanding of the action of estrogen and progesterone involves the effects of these hormones within the brain , but they also affect many parts of the body that in turn alter brain activity . this complicated interplay between the brain and the rest of the body is one of the reasons epilepsy is hard to treat it may not be a static condition but one that is constantly changing . several hypotheses based on effects of estrogen and progesterone that have been identified in the last decades have been offered to explain catamenial epilepsy . one is that seizures increase when the ratio of estrogen to progesterone is high.5,19,20 this hypothesis explains the worsening of seizures during the periovulatory period and the relative resistance to seizures during the luteal phase ( days 1428see diagram 1 , page 9 ) , but it does not explain why seizures worsens during the perimenstrual period , because both estrogen and progesterone are low at that time . an alternative view developed after it was shown that falling progesterone levels exert excitatory effects.21 researchers hypothesized that progesterone withdrawal occurs at the end of the menstrual cycle , leading to excitatory effects . literally , there is a decrease in seizure threshold,22 which is the point at which the brain has been sufficiently activated so that a seizure begins . in patients with epilepsy , this threshold is thought to be lower than in other people , so that even mild activation of the brain can lead to a seizure . researchers investigated the notion of progesterone withdrawal by treating laboratory rats or mice with progesterone and then suddenly stopping the treatment . the results showed that a decrease in seizure threshold accompanied withdrawal of the treatment.21,22 these data suggest that in a patient with epilepsy , seizures may be more likely after progesterone withdrawal because seizure threshold is lower . subsequent studies , using other methods to simulate progesterone withdrawal , and other means of evaluating seizure susceptibility , confirmed these findings.23,24 progesterone withdrawal therefore provides a potential explanation for perimenstrual seizures . further research into the effects of progesterone withdrawal has suggested that it is not simply a sudden drop in progesterone or its metabolite allopregnanolone that causes the increased susceptibility to seizures . rather , progesterone withdrawal leads to a specific change in gabaa receptors , the neurotransmitter receptors that bind allopregnanolone . the gabaa receptor is normally composed of several components , or subunits . when progesterone withdrawal occurs , increased levels of one of the subunits stops allopregnanolone from having an inhibitory effect on seizure activity,25,26 and other subunit changes lead to the resistance of seizures to some antiepileptic drugs , such as the benzodiazepines.27 progesterone also affects other hormones , called mineralocorticoids . mineralocorticoids normally allow the body to retain sodium , potassium , and , as a result , water . this is significant because water balance affects seizures : when neurons in the brain swell , they become closer together , increasing neuronal activity.28 as a result , seizure threshold can decrease . during the menstrual cycle , water retention occurs during the end of the luteal phase . during this period , which coincides with the perimenstrual phase , seizure threshold may decrease because water retention increases neuronal excitability . this reasoning has led to the use of diuretics such as acetazolamide in women with catamenial epilepsy , although they have not been completely successful,29 probably because they do not treat the other effects of hormones during the menstrual cycle . recent studies in female laboratory animals suggest that estrogen levels may also contribute to perimenstrual seizures , because estrogen can have effects on the perimenstrual period even if estrogen is elevated only in the preceding phases of the cycle . for example , estrogen increases the concentration of brain - derived neurotrophic factor ( bdnf ) , a molecule that increases excitation in the brain . the increase in bdnf is long - lasting it persists after estrogen levels have returned to normal.30 estrogen can therefore exert long - lasting effects on neuronal activity even if estrogen levels increase only briefly . high levels of bdnf throughout the luteal phase would result ; this prediction is supported by clinical studies showing that bdnf is high during the luteal phase.31 during the luteal phase , the actions of bdnf may be inhibited by progesterone and allopregnanolone , but at the end of the menstrual cycle , elevated bdnf may persist after progesterone and allopregnanolone fall . the imbalance between excitatory effects of bdnf and inhibitory effects of progesterone and allopregananolone would explain increased seizures during the perimenstrual period . it is also interesting that bdnf induces the synthesis of neuropeptide y ( npy ) , which is considered to be a potential anticonvulsant.32 the rise in npy could truncate the effects of bdnf , ending the perimenstrual period . there are many neurological and psychiatric conditions in which symptoms worsen in relation to the menstrual cycle . some scientists have suggested that the symptoms are caused by abnormal periods of activity in parts of the central nervous system ( cns ) , and resemble seizures . with regard to migraine headaches , for example , women often report that the most severe episodes follow a perimenstrual pattern . and during a migraine , abnormal patterns of brain activity develop . women who have chronic pain can experience the most severe pain during certain phases of the menstrual cycle . pain may result from a period of hyperactivity in the areas of the cns where pain is controlled . the concept of an epilepsy spectrum suggests that these types of hyperactivity of neurons in the cns might be a part of the spectrum , even though pain has not been considered to be a type of epilepsy . supporting this view is the observation that antiepileptic drugs are often successful in the treatment of migraine and pain . why do only some women have catamenial epilepsy , and why , even among those women , is it sometimes difficult to document ? the type of epilepsy that is most susceptible to a catamenial pattern appears to involve a part of the brain ( the temporal lobe ) that does not control movement , so the seizures may not be convulsive.33 there are a variety of nonconvulsive seizures , and they have different causes . different areas of the brain are involved . in temporal lobe epilepsy , for example , seizures are often partial or complex partial : they can arise from one part of the temporal lobe and spread to other areas ( but do not always do so ) , and they are not always accompanied by convulsions . these seizures often start with an aura a sensation of dj vu , for example , or an awareness of a familiar smell and are usually accompanied by a temporary loss of consciousness . after the seizure , the person may be somewhat confused but subsequently resumes normal behavior . because a patient with temporal lobe epilepsy can have seizures without convulsions , the person can be unaware that a seizure occurred he or she would have no memory of it . the person may also not remember the seizure because of the disruption of activity in the temporal lobe at the time of the seizure the temporal lobe is important to normal learning and memory . it is easy to see that the prevalence of catamenial epilepsy could well be underestimated if the type of seizure activity that increases at particular times during the menstrual cycle is nonconvulsive . the occurrence of catamenial epilepsy may also be underestimated because patients may experience nonconvulsive seizures in a catamenial pattern but their physicians do not know they occurred . in addition , physicians can not be sure that all seizures patients report actually occur , because some people appear to imagine seizures these are called pseudoseizures.34 most physicians would use an electroencephalogram ( eeg ) to document seizures , but some seizures may be missed by eeg if they involve structures deep in the brain , relatively far from the eeg electrodes , which are placed on the scalp , near the brain s surface . may not be diagnosed with catamenial epilepsy if their menstrual cycle is not regular . because chronic seizures can lead to irregular cycles , a woman and her physician may be unable to recognize a pattern . the notion of a spectrum of epilepsy is not only important to women with epilepsy but relevant to research scientists who use laboratory animals to study temporal lobe epilepsy . in these animals , seizures that simulate those in patients with temporal lobe epilepsy are usually defined by five stages of convulsive seizures that were originally described in 1972.35 the stages are easy to see because there are convulsions . it is hard for researchers to agree when an animal has a nonconvulsive seizure , in part because of the difficulty in defining the point at which normal brain activity has increased enough to constitute a seizure . by considering epilepsy as a spectrum ranging from mild to severe , and no longer requiring convulsions to define a seizure , research in epilepsy would broaden in scope , and ultimately many more people with seizures would be helped , such as those people who have seizures that are hard to detect . the more researchers can tell us about the spectrum of seizures in experimental animals , the better equipped we will be to treat the spectrum of seizure disorders that exist in people . in fact , some potential antiepileptic drugs may have been passed over in the last decades of drug screening because drugs are usually tested to see if they stop convulsive seizures in animals . and they are often not used to see if they help animals that are tested for other conditions where nonconvulsive seizures are suspected , such as alzheimer s disease . some candidate antiepileptic drugs might be successful against nonconvulsive conditions but would fail a drug screen because they are unable to ameliorate a convulsive seizure . restricting epilepsy research to the investigation of convulsive seizures is especially limiting for studies of hormones in female animals because convulsive seizures in female rodents stop the ovarian cycle or make it irregular . this effect has been documented for the common animal models of temporal lobe epilepsy.3638 as a result , it is hard to conduct research in female rodents to understand how epilepsy influences reproductive function . this is unfortunate , because women with epilepsy often develop reproductive problems , such as infertility or polycystic ovarian syndrome . studying female rats with nonconvulsive seizures would provide more opportunities to understand the adverse effects of seizures on the reproductive system . the idea of the epilepsy spectrum also has implications for the development of new drugs to treat epilepsy . it may be useful to develop drugs that block the consequences of estrogen and progesterone , rather than estrogen and progesterone themselves , because the hormones are very important to normal life . if a particular branch of bdnf signaling could be identified as the most important to seizure threshold , it might be a better endpoint to target . this strategy could be used in concert with progesterone supplementation , or the enhancement of allopregnanolone levels . however , bdnf signaling and gabaa receptors are important to many normal functions . more specific targets than bdnf or the gabaa receptor are needed so that anticonvulsant actions occur without side effects . the more we can clarify the mechanisms underlying catamenial seizure exacerbation , the more likely it is that we can develop treatments without side effects . epilepsy is a devastating illness that has far - reaching effects on patients and their families . patients could be better helped by greater awareness of the epilepsy spectrum because nonconvulsive seizures would be better recognized . deepening the understanding of the epilepsy spectrum could lead to more accurate diagnoses , possibly allowing the detection of epilepsy in individuals who would otherwise not be treated with anticonvulsants . research would benefit by the increasing recognition of the diverse types of seizures , which would lead to a broader focus on drugs that are not necessarily anticonvulsant but antiepileptic .
integration of the reverse - transcribed viral dna into the host genome is an essential step in the lifecycle of retroviruses . retrovirus integrase ( in ) catalyzes insertions of both ends of the linear viral dna into a host chromosome 1 . in from hiv-1 and closely related retroviruses share the three - domain organization , consisting of a catalytic core domain flanked by n- and c - terminal domains essential for the concerted integration reaction . although structures of the tetrameric in - dna complexes have been reported for in from prototype foamy virus ( pfv ) featuring an additional dna - binding domain and longer interdomain linkers 25 , the architecture of a canonical three - domain in bound to dna remained elusive . here we report a crystal structure of the three - domain in from rous sarcoma virus ( rsv ) in complex with viral and target dnas . the structure shows an octameric assembly of in , in which a pair of in dimers engage viral dna ends for catalysis while another pair of non - catalytic in dimers bridge between the two viral dna molecules and help capture target dna . the individual domains of the eight in molecules play varying roles to hold the complex together , making an extensive network of protein - dna and protein - protein contacts that show both conserved and distinct features compared to those observed for pfv in . our work highlights diversity of retrovirus intasome assembly and provides insights into the mechanisms of integration by hiv-1 and related retroviruses .	rsv in and its mutant forms were overexpressed in escherichia coli bl21 ( de3 ) and purified as previously described . the proteins were stored in aliquots at 80c in 20 mm hepes - naoh ( ph 7.5 ) , 1.0 m nacl , 20m zncl2 , 5 mm 2-mercaptoethanol and 10% ( w / v ) glycerol . the branched dna substrate mimicking the product of the concerted integration reaction was obtained by annealing three synthetic oligonucleotides ( integrated dna technologies ) . a similar strategy had been used previously to prepare a pfv in - dna complex , which demonstrated that the in - dna complex assembled on the designed integration product has essentially an identical structure to the equivalent complex formed via the forward integration reaction . the viral dna branches carrying the high - affinity gain - of - function mutant rsv u3 long terminal repeat ( ltr ) sequence ( gu3 ) were attached to the target dna duplex with a palindromic 6 bp spacer to generate a fully symmetrized structure . to prepare the rsv in - dna complexes for crystallization , 30 m half - site dna substrate was mixed with 120 m rsv in in 20 mm hepes - naoh ( ph 7.5 ) , 500 mm nacl , 20% ( w / v ) glycerol , and 1 mm tris-(2-carboxyethyl)phosphine ( tcep ) . the mixture was dialyzed against low salt buffer ( 20 mm hepes - naoh ph 7.5 , 125 mm nacl , 20% ( w / v ) glycerol , 1 mm tcep ) at 25c for overnight . at the end of this first dialysis , the mixture was subsequently dialyzed against high salt buffer ( 20 mm mes - naoh ph 6.0 , 1.2 m nacl , 20% ( v / v ) dimethyl sulfoxide ( dmso ) , 5% ( w / v ) glycerol , 1 mm tcep ) at 25c for 24 hours . at the end of this second dialysis , the solubilized rsv in - dna complex ( intasome ) was purified through size - exclusion chromatography ( superdex 200 10/300 gl , ge healthcare ) running with 20 mm mes - naoh ph 6.0 , 1.2 m nacl , 20% ( v / v ) dmso , 5% ( w / v ) glycerol , and 1 mm tcep . the isolated intasome remains stable in the high - salt condition containing 1.2 m nacl that precludes complex formation , suggesting that once the intasome forms , it is kinetically trapped . the solubility - enhancing rsv in mutation f199k completely abolished intasome formation . for the sec - mals analysis , a modified condition was used for the intasome re - solubilization and isolation for better baseline stability ( extended data fig . the rsv intasome assembled through dialysis and purified by size - exclusion chromatography was concentrated to 46 mg / ml using centrifugal concentrator ( amicon ) . various combinations of the lengths of the viral dna ( ranging from 16 bp to 25 bp ) and flanking target dna ( ranging from 14 bp to 22 bp , corresponding to the full target dna lengths of 34 bp to 50 bp ) were screened in crystallization trials . the extensive screening yielded only one crystal form with a specific combination of 22-base ( length of the non - transferred strand ) viral dna branches and 16 base - pair ( bp ) target dna flanks on either side of the central six bp spacer ( fig . dna substrates with two slightly different target sequences were used ( 5-aatgttgtcttatgcaatactc-3/5-gagtattgcataagacaacagtgcacgaatcttgaagacact-3/5-agtgtcttcaagattc-3 , or 5-aatgttgtcttatgcaatactc-3/5-gagtattgcataagacaacagtcgaccaaccttcaacttagc-3/5-gctaagttgaaggttg-3 ) , and they produced essentially the same crystals . the rsv intasome crystals were grown through reverse vapor diffusion in hanging drops at 22c , by mixing 1.5 l in - dna complex solution with 1.5 l reservoir solution ( 3.2 m sodium formate ) . crystals appeared within 35 days and reached a size of ~150300 m in 3~5 days . even though the rsv intasome crystals initially diffracted x - ray poorly ( ~ 10 ) , soaking the crystals with a metatungstate cluster compound dramatically improved the resolution ( extended data fig . the tungsten cluster was later found to bind between in dimers from separate intasome complexes to mitigate crystal lattice disorder . the crystals were soaked overnight with 0.15 mm metatungstate cluster na6[h2w12o40 ] in a stabilization buffer consisting of 3.2 m sodium formate , 16 mm mes - naoh ph 6.0 , 0.8 m nacl , 16% ( v / v ) dmso , 4% ( w / v ) glycerol , and 1 mm tcep . after soaking , the crystals were cryo - protected in 3.2 m sodium formate , 16 mm mes ph 6.0 , 0.8 m nacl , 16% ( v / v ) dmso , 12% ( w / v ) glycerol , and 1 mm tcep , and were frozen by rapid immersion in liquid nitrogen . the full - length wild - type rsv in(1286 ) , the c - terminally truncated rsv in(1270 ) , and its various mutant forms tested produced essentially the same crystals with indistinguishable x - ray diffraction properties . x - ray diffraction data were collected at the advanced photon source northeastern collaborative access team beamlines ( 24-id - c / e ) and the advanced light source molecular biology consortium ( 4.2.2 ) beamline and processed using hkl2000 or xds . the rsv intasome crystals showed varied degrees of pseudo - merohedral twinning with twin operator [ l , k , h ] , owing to the very similar a and c unit cell dimensions of the primitive monoclinic lattice . thus , we screened a large number of crystals to identify ones that diffract to higher resolution and have smaller twin fractions . the structure of rsv intasome was determined by molecular replacement with phaser , using the rsv in ccd , ctd ( pdb i d : 4fw1 ) , and a 16 bp b - form dna as search models . 8 copies of ccd , one copy of ctd and three copies of dna molecules were located . refinement of the partial model revealed electron density for two copies of the metatungstate clusters . subsequent iterative model building using coot and refinement with phenix suite allowed placement of the remaining 7 copies of ctd , 8 copies of ntd generated using modeller based on hiv-1 ntd ( pdb i d : 1k6y ) , and building of the inter - domain linkers as well as the remaining parts of the dna molecule guided by the difference electron density maps . a third metatungstate cluster , which is more weakly bound compared to the first two , was positioned manually into residual density . the dna base - pairs and base - stacking restraints were used throughout the refinement . the geometry restraints for protein included the reference - model restraints for ccd and ctd based on the higher resolution rsv in structure ( 4fw1 ) , and the secondary structure and zinc - coordination restraints for ntd . atomic displacement parameters were refined with grouped b - factors per residue for protein and dna , and a total of 53 tls groups assigned by phenix . the asymmetric unit of the crystal contains one complete rsv intasome , which includes eight in molecules and two viral dna branches emanating from a strongly bent 38 bp target dna . the dataset used for the final refinement was from an rsv intasome crystal grown using selenomethionine - labeled rsv in ( 1270 ) with the following amino acid substitutions ; c23s , l112 m , l135 m , l162 m , l163 m , l188 m , and l189 m , which we confirmed to be active in concerted integration and inhibited by insti similarly to the wild - type rsv in , and a dna substrate carrying a nick at the middle of each target dna branch ( the 16-base dna strand shown in olive in fig . the nick occasionally facilitated crystal growth , but it was not necessary for crystallization and did not change the space group or the unit cell parameters compared to crystals grown without the nick . because this nick in the target dna is not biologically relevant , it is not shown in figs . twin refinement protocol was not used as the dataset used for the final refinement had a low ( less than 10% ) twin fraction . the summary of data collection and refinement statistics is shown in extended data table 1 . the paired - refinement procedure was performed in steps of 0.1 to determine the high - resolution limit ( extended data fig . the register of amino acids in the final model was verified by the selenium anomalous difference fourier peaks ( extended data fig . ramachandran analysis shows that 96.0 , 3.9 , and 0.1 % of the protein residues are in the most favored , allowed , and disallowed region , respectively . the ntd - ccd linker in some of the non - catalytic in molecules and the last four bp at the distal end of one of the viral dna molecules were not built due to poor electron density . rsv in and its mutant forms were overexpressed in escherichia coli bl21 ( de3 ) and purified as previously described . the proteins were stored in aliquots at 80c in 20 mm hepes - naoh ( ph 7.5 ) , 1.0 m nacl , 20m zncl2 , 5 mm 2-mercaptoethanol and 10% ( w / v ) glycerol . the branched dna substrate mimicking the product of the concerted integration reaction was obtained by annealing three synthetic oligonucleotides ( integrated dna technologies ) . a similar strategy had been used previously to prepare a pfv in - dna complex , which demonstrated that the in - dna complex assembled on the designed integration product has essentially an identical structure to the equivalent complex formed via the forward integration reaction . the viral dna branches carrying the high - affinity gain - of - function mutant rsv u3 long terminal repeat ( ltr ) sequence ( gu3 ) were attached to the target dna duplex with a palindromic 6 bp spacer to generate a fully symmetrized structure . to prepare the rsv in - dna complexes for crystallization , 30 m half - site dna substrate was mixed with 120 m rsv in in 20 mm hepes - naoh ( ph 7.5 ) , 500 mm nacl , 20% ( w / v ) glycerol , and 1 mm tris-(2-carboxyethyl)phosphine ( tcep ) . the mixture was dialyzed against low salt buffer ( 20 mm hepes - naoh ph 7.5 , 125 mm nacl , 20% ( w / v ) glycerol , 1 mm tcep ) at 25c for overnight . at the end of this first dialysis , the mixture was subsequently dialyzed against high salt buffer ( 20 mm mes - naoh ph 6.0 , 1.2 m nacl , 20% ( v / v ) dimethyl sulfoxide ( dmso ) , 5% ( w / v ) glycerol , 1 mm tcep ) at 25c for 24 hours . at the end of this second dialysis , the solubilized rsv in - dna complex ( intasome ) was purified through size - exclusion chromatography ( superdex 200 10/300 gl , ge healthcare ) running with 20 mm mes - naoh ph 6.0 , 1.2 m nacl , 20% ( v / v ) dmso , 5% ( w / v ) glycerol , and 1 mm tcep . the isolated intasome remains stable in the high - salt condition containing 1.2 m nacl that precludes complex formation , suggesting that once the intasome forms , it is kinetically trapped . the solubility - enhancing rsv in mutation f199k completely abolished intasome formation . for the sec - mals analysis , a modified condition was used for the intasome re - solubilization and isolation for better baseline stability ( extended data fig . for crystallization , the rsv intasome assembled through dialysis and purified by size - exclusion chromatography was concentrated to 46 mg / ml using centrifugal concentrator ( amicon ) . various combinations of the lengths of the viral dna ( ranging from 16 bp to 25 bp ) and flanking target dna ( ranging from 14 bp to 22 bp , corresponding to the full target dna lengths of 34 bp to 50 bp ) were screened in crystallization trials . the extensive screening yielded only one crystal form with a specific combination of 22-base ( length of the non - transferred strand ) viral dna branches and 16 base - pair ( bp ) target dna flanks on either side of the central six bp spacer ( fig . dna substrates with two slightly different target sequences were used ( 5-aatgttgtcttatgcaatactc-3/5-gagtattgcataagacaacagtgcacgaatcttgaagacact-3/5-agtgtcttcaagattc-3 , or 5-aatgttgtcttatgcaatactc-3/5-gagtattgcataagacaacagtcgaccaaccttcaacttagc-3/5-gctaagttgaaggttg-3 ) , and they produced essentially the same crystals . the rsv intasome crystals were grown through reverse vapor diffusion in hanging drops at 22c , by mixing 1.5 l in - dna complex solution with 1.5 l reservoir solution ( 3.2 m sodium formate ) . crystals appeared within 35 days and reached a size of ~150300 m in 3~5 days . even though the rsv intasome crystals initially diffracted x - ray poorly ( ~ 10 ) , soaking the crystals with a metatungstate cluster compound dramatically improved the resolution ( extended data fig . the tungsten cluster was later found to bind between in dimers from separate intasome complexes to mitigate crystal lattice disorder . the crystals were soaked overnight with 0.15 mm metatungstate cluster na6[h2w12o40 ] in a stabilization buffer consisting of 3.2 m sodium formate , 16 mm mes - naoh ph 6.0 , 0.8 m nacl , 16% ( v / v ) dmso , 4% ( w / v ) glycerol , and 1 mm tcep . after soaking , the crystals were cryo - protected in 3.2 m sodium formate , 16 mm mes ph 6.0 , 0.8 m nacl , 16% ( v / v ) dmso , 12% ( w / v ) glycerol , and 1 mm tcep , and were frozen by rapid immersion in liquid nitrogen . the full - length wild - type rsv in(1286 ) , the c - terminally truncated rsv in(1270 ) , and its various mutant forms tested produced essentially the same crystals with indistinguishable x - ray diffraction properties . x - ray diffraction data were collected at the advanced photon source northeastern collaborative access team beamlines ( 24-id - c / e ) and the advanced light source molecular biology consortium ( 4.2.2 ) beamline and processed using hkl2000 or xds . the rsv intasome crystals showed varied degrees of pseudo - merohedral twinning with twin operator [ l , k , h ] , owing to the very similar a and c unit cell dimensions of the primitive monoclinic lattice . thus , we screened a large number of crystals to identify ones that diffract to higher resolution and have smaller twin fractions . the structure of rsv intasome was determined by molecular replacement with phaser , using the rsv in ccd , ctd ( pdb i d : 4fw1 ) , and a 16 bp b - form dna as search models . 8 copies of ccd , one copy of ctd and three copies of dna molecules were located . refinement of the partial model revealed electron density for two copies of the metatungstate clusters . subsequent iterative model building using coot and refinement with phenix suite allowed placement of the remaining 7 copies of ctd , 8 copies of ntd generated using modeller based on hiv-1 ntd ( pdb i d : 1k6y ) , and building of the inter - domain linkers as well as the remaining parts of the dna molecule guided by the difference electron density maps . a third metatungstate cluster , which is more weakly bound compared to the first two , was positioned manually into residual density . the dna base - pairs and base - stacking restraints were used throughout the refinement . the geometry restraints for protein included the reference - model restraints for ccd and ctd based on the higher resolution rsv in structure ( 4fw1 ) , and the secondary structure and zinc - coordination restraints for ntd . atomic displacement parameters were refined with grouped b - factors per residue for protein and dna , and a total of 53 tls groups assigned by phenix . the asymmetric unit of the crystal contains one complete rsv intasome , which includes eight in molecules and two viral dna branches emanating from a strongly bent 38 bp target dna . the dataset used for the final refinement was from an rsv intasome crystal grown using selenomethionine - labeled rsv in ( 1270 ) with the following amino acid substitutions ; c23s , l112 m , l135 m , l162 m , l163 m , l188 m , and l189 m , which we confirmed to be active in concerted integration and inhibited by insti similarly to the wild - type rsv in , and a dna substrate carrying a nick at the middle of each target dna branch ( the 16-base dna strand shown in olive in fig . the nick occasionally facilitated crystal growth , but it was not necessary for crystallization and did not change the space group or the unit cell parameters compared to crystals grown without the nick . because this nick in the target dna is not biologically relevant , it is not shown in figs . twin refinement protocol was not used as the dataset used for the final refinement had a low ( less than 10% ) twin fraction . the summary of data collection and refinement statistics is shown in extended data table 1 . the paired - refinement procedure was performed in steps of 0.1 to determine the high - resolution limit ( extended data fig . the register of amino acids in the final model was verified by the selenium anomalous difference fourier peaks ( extended data fig . ramachandran analysis shows that 96.0 , 3.9 , and 0.1 % of the protein residues are in the most favored , allowed , and disallowed region , respectively . the ntd - ccd linker in some of the non - catalytic in molecules and the last four bp at the distal end of one of the viral dna molecules were not built due to poor electron density . the secondary structure elements for rsv in are color - coded based on the three in domains similarly to fig . a , the half - site ( gaped duplex ) substrate prepared by annealing three oligonucleotides dimerizes via the self - complementary 6-base spacer sequence ( underlined ) to form a branched structure mimicking the product of the concerted integration reaction ( fig . viral dna nucleotides are numbered , and some of the structural elements of rsv in involved in the viral dna interactions are shown . c , x - ray diffraction pattern after the metatungstate - soaking ( see method for details ) . is perpendicular to the two - fold screw ( b ) axis of the monoclinic lattice , which lies horizontally . f , paired - refinement analysis to assess the resolution limit of the rsv intasome diffraction data . for each pairwise comparison , model refinements were run at two different resolution limits and the r - factors calculated for a common ( lower ) resolution cutoff were compared . a , a representative size - exclusion chromatography ( sec ) profile for rsv intasome , overlaid with that for a mixture of molecular weight markers . b , sec profiles for rsv intasomes formed with in of varying c - termini . c , sec - mals ( multi - angle light scattering ) analysis of rsv intasome . the intasome formed with rsv in ( 1269 aa ) was separated by sec in a modified condition containing 20 mm hepes ph 7.5 , 1.0 m nacl , 5 % glycerol , and 1.0 mm tcep . the absolute molecular mass was determined by light scattering using in - line detectors described previously . a similar sec - mals analysis of the intasome formed with wt full - length rsv in ( 1286 aa ) yielded a molecular mass of 268 kda ( n=2 , data not shown ) . the calculated mass of an intasome containing eight rsv in ( 1269 or 270 ) molecules is ~288 kda . d , chemical cross - linking analysis of rsv intasome . the rsv intasome and free in ( 1269 aa ) were purified by size - exclusion chromatography in the running buffer : 20 mm hepes ( ph 7.5 ) , 1.0 m nacl , 5 % glycerol , and 1.0 mm tcep . the peak fractions of the intasome and in were cross - linked with the indicated amount of ethylene glycol bis - succinimidylsuccinate ( egs ) as described previously and analyzed by sds - page . the majority of cross - linked species within the intasome were larger than a tetramer . the nupage 412% gradient gel with a mes - based sds - page running buffer was used . anomalous difference fourier maps calculated using the data collected on selemomethionine - labeled rsv intasome , contoured at 3.5 ( blue mesh ) or 5.0 ( orange mesh ) . b , c , close - up view of a proximal ( b ) and distal ( c ) in dimer , respectively . a , b , protein arrangement in the octameric rsv intasome . the inner and outer subunit of one proximal in dimer is colored in green and cyan , respectively , with the catalytic triad ( dde ) of the inner subunit shown in red . c , a close - up view around the active site of the inner in subunit in the rsv intasome . the dna strands are colored as in fig . 1 , and the catalytic triad residues ( dde ) the color scheme follows that used for the proximal in dimers of rsv in in ( a , b ) . f , a close - up view around the active site of the inner in subunit in the pfv intasome ( pdb i d : 3os0 ) . simulated annealing composite omit 2mfodfc density contoured at 1.0 , shown for area within 3.5 from any protein or dna atom in the final model . in a and b , electron densities around protein and dna are colored differently ( blue and green , respectively ) . a , b , dna structure in the rsv ( a ) or pfv ( b ) intasome , alternatively referred to as the strand - transfer complex ( stc ) . c , a comparison of dna structures between the rsv and pfv intasomes ( stcs ) . the integration product dnas ( rsv in cyan , pfv in red ) superimposed at a viral dna terminus are shown in three different view angles . note the significant deviation in overall trajectory of the target dna , and difference in the orientation of the second viral dna molecule . the region spanning the two integration sites on opposing target dna strands is 6 bp for rsv and 4 bp for pfv . the sigma a - weighted 2mfodfc map contoured at 1.5 ( a ) or 2.5 ( b ) , overlaid with the final model for the central 6 bp region between the two integration sites . data collection and refinement statistics statistics for the highest resolution shell are shown in parentheses . a video showing the overall structure of the rsv intasome and positioning of the three structural domains of in within the intasome .
a case of tick bite was found in the inguinal region of a 74-year - old korean woman . she was attacked by the tick while working in her vegetable garden in the vicinity of mountain located in suncheon city , the southern coastal area of the korean peninsula . on admission she complained of mild discomfort and itching around the bite area . the causative tick was 23 mm long and had slender pedipalps . the scutum was quite ornate and had eyes at the edge . the genital aperture was located anterior to the level of the coxa ii . the spiracular plate was comma - shaped and the anus was surrounded posteriorly by the anal groove . the coxa i had subequal 2 spurs ; the external one slightly larger . the spur of coxa iv was slightly longer than those of coxae ii and iii . the tarsus iv had 2 distinct subapical ventral spurs . it was identified as the fully engorged adult female of amblyomma testudinarium . this is the first human case of amblyomma bite in korea .	a lot of species are vectors of viral , rickettsial , bacterial , and protozoal diseases of birds and mammals , including humans . identification of causative tick is important in making a diagnosis in tick - related dermatoses . in the republic of korea , hard ticks which belong to the family ixodidae are responsible for most tick - related diseases . since the first human case of tick bite was reported in 1982 , the total number of cases recorded in the literature reached approximate 40 . no amblyomma case has yet been recorded in korea although approximately 100 species of the genus amblyomma were recorded worldwide . here , we report a human case of typical infestation with an amblyomma testudinarium ( koch , 1844 ) adult worm in a korean woman . in july 2009 , a 74-year - old korean woman presented to our dermatology clinic with a large mass on her left inguinal region . she was a typical rural housewife frequently worked in the field near her house . on admission she complained of mild discomfort and itching sensation of the skin around the bite area . laboratory examinations were normal or negative for the following tests : hemoglobin 14.1 g / dl , hematocrit 43.3% , white blood cell 8,210/mm ( neutrophil 69% , lymphocyte 25% , monocyte 6% ) , erythrocyte sedimentation rate 6 mm / hr , serum glutamic pyruvic transaminase 30 ten days prior to presentation , the patient began to note the presence of a mass which she thought to be benign . in the clinic , we found a dark green , round , 23 mm organism whose head was partially burrowed under the skin . it was grasped close to the skin and steady pressure applied , pulling the tick straight out perpendicularly to the skin . when the organism was removed by plucking , its mouth part was torn . six months later , the patient was well and then follow - up communication was discontinued . she was probably attacked by the tick while working to collect vegetables and edible sprouts of wild grass in her vegetable garden located in the suburb of suncheon city , jeollanam - do , where she had been residing . she recently had not traveled to any foreign country as well as any domestic area , including jeju island . the isolated tick was fixed with formalin and then stereoscopically examined to determine its species . the body was 23 mm in length , 19 mm in width , and 12 mm in height ( fig . the small ornamented scutum was seen as a small shield behind the capitulum on the back . the eyes were located on the lateral edges of the scutum but not projecting beyond scutal contour . although greater part of the mouth including the hypostome was torn off , the left pedipalp only was relatively well preserved and had characteristic appearance ; especially long article 2 with approximately 2.5 times as long as article 3 ( fig . the genital aperture was located at the level between coxa i and coxa ii ( figs . 1 , 2b ) . the anus was located at the level posterior to the spiracular plates and embraced by y - shaped anal groove posteriorly ( figs . 1 , 2c ) . the spiracular plate , comma - shaped , was located on the ventrolateral surface posterolaterally to the coxa iv ( figs . 1 , 2d ) . the tick had 4 pairs of legs ( figs . 1 , 3 ) . the coxa i had 2 medium sized subequal spurs , external spur was slightly longer than internal spur ( fig . the coxa ii had a single salient ridgelike spur which was much broader than long ( fig . the coxa iii with a single spur was very similar to coxa ii ( fig . the coxa iv had a single spur which was longer than those of the coxae ii and iii ( fig . base upon these morphological features , this tick was identified as a female adult of a. testudinarium ( table 1 ) . ticks are blood - sucking arthropods that parasitize various species of vertebrates in almost every region of the world . in the republic of korea , about 40 human cases have been reported since 1982 [ 3 - 5 ] . the causative ticks reported in korea were identified as ixodes nipponensis , i. ovatus , i. monospinosus , i. persulcatus , haemaphysalis flava , and h. longicornis . the genus amblyomma ticks are large and beautifully ornamented with long mouth parts ; possessing eyes and festoons . close examination under a stereomicroscope showed the capitulum of the anterior portion of the body , the scutum of the dorsal portion , and the spiracular plate , genital aperture , anus , and 4 pairs of legs of ventral portion . the pedipalp of the capitulum was slender with the second segment much longer than the third . the scutum was quite ornate in comparison to other parts of the body and had non - protruding eyes at the edge . the coxa of the first leg had subequal 2 spurs ( the external one was slightly larger than internal ) . the spine of coxa on the fourth leg was longer than those on the second and third legs . these characteristics were typical features of the adult female of a. testudinarium [ 8 - 11 ] . they feed for 7 to 12 days for a full engorgement and reach 20 mm in length after feeding . the genus amblyomma is one of the largest ticks among the hard ticks . in case of a. testudinarium , the size of adult females varies from 5 to 20 mm depending on the stages of engorgement . however , this species may feed for 30 days or longer and reach bigger than 20 mm in length . in the present case , hospital admission was quite delayed . the tick fed over additional 10 days after detected by the patient and was inordinately engorged . it measured 23 mm long , which was bigger than any other human ticks reported in korea . the present tick would have been in situ for at least 1 month . during that time , the patient had some itching and mild pain of the skin around the bite area , but no clinical symptoms of tick - borne infection , including lyme disease and rickettsiosis , occurred . ticks can also cause inflammatory problems in the host without transmitting infection . when the head part was broken off in the removal process , more careful attention her symptoms disappeared gradually after the removal of the tick and the surgical wound was healed in a week . the frequent location involved by a. testudinarium is known to be the axillary , anogenital , or inguinal region , which are rich in apocrine glands . in the present case , tick bite was found on the inguinal region . according to the patient 's recall of daily life , she often undressed for urination while working at the vegetable garden nearby her home . it appears that the tick attached itself to her clothing or skin when she was undressed . a. testudinarium is known to be a tropical tick and found mainly in the indian peninsula , south east asia , including myanmar , thailand , malaysia , indonesia , the philippines , taiwan , and japan . in japan , it is especially important in southwestern areas ( below about 36 north latitude ) , including kinki , shikoku , kyushu , and chugoku . the geographical limitation of a. testudinarium in the southwestern part of japan implies that the warm temperature with heavy rainfall and the subtropical vegetation fauna may be a proper environmental condition for this tick . in korea , a. testudinarium has been known to be distributed in jeju island ( between 33 to 34 north latitude ) , which is located in the south sea far from the korean peninsula . however , this species was also collected in a southern coastal area of jeollanam - do during a haemaphysalis phasiana survey using tick drags . in the present case , the patient has been living in a suburban or rural area of suncheon city , approximately 400 km south of seoul , korea . it is an agricultural and industrial city nearby the sea at suncheon bay ( 35 north latitude ) facing the south sea .
hashimoto 's encephalopathy ( he ) is a rare steroid - responsive encephalopathy syndrome , which can have highly variable neuropsychiatric manifestations and can go unrecognized for a long time . he is a diagnosis of exclusion and should be kept in mind when evaluating a patient with a cognitive dysfunction and high titers of anti - thyroid antibodies as it responds dramatically to steroids . steroid responsive myoclonus can be a presentation of he .	hashimoto 's encephalopathy is a rare steroid responsive encephalopathy syndrome characterized by persisting or fluctuating neurologic and neuropsychological deficits associated with elevated blood concentrations of anti - thyroid antibodies . the classically described clinical manifestations usually include acute to subacute onset of confusion with alteration of consciousness , but myoclonus is a less publicized manifestation of the disease . we present a case of hashimoto 's encephalopathy , presenting as steroid responsive myoclonus in a setting of autoimmune thyroid disease . an 18-year - old male patient was diagnosed to have primary hypothyroidism 6 months back following weight gain , edema and facial puffiness of 3 years duration . his initial thyroid function test ( tft ) was t4 : 2.3 g / dl ( 5.01 - 12.4 ) t3 : 0.35 ng / ml ( 0.6 - 1.81 ) and thyroid stimulating hormone ( tsh ) : 180 iu / ml ( 0.35 - 5.50 ) and was started on l thyroxine , with a significant improvement of symptoms . he started developing myoclonus for the past 1 month , for which he was referred to our hospital . on examination , he was conscious , with a cognitive impairment and had memory loss for recent events , with the poor attention span , difficulty in finding words , dyscalculia and dysgraphia . his mini mental status examination ( mmse ) was 11 suggestive of moderate cognitive impairment . he also had myoclonus , involving both upper limbs and lower limbs . on investigations , blood routine examination and erythrocyte sedimentation rate was normal and liver function test , kidney function test , sodium , potassium , calcium and magnesium were normal and so were the arterial blood gas analysis . his latest tfts show normal t3 and t4 with mild elevation of tsh ( tsh-9.1 iu / ml ) . his electroencephalogram ( eeg ) showed a diffuse slow wave activity [ figure 1 ] and magnetic resonance images ( mri ) showed a pituitary hyperplasia [ figure 2 ] with non - specific white matter changes [ figure 3 ] . electroencephalogram showing diffuse slow wave activity magnetic resonance images showing pituitary hyperplasia magnetic resonance images showing non - specific white matter changes antinuclear antibody titer , anti - double - stranded deoxyribonucleic acid , hepatitis b surface antigen , anti - hepatitis c virus , lupus anticoagulant and venereal disease research laboratory ( vdrl ) test were carried out to evaluate the cause of decreased cognitive function , myoclonus and seizures , which were all negative . csf electrophoresis was normal . in view of these neurologic symptoms , associated with high titers of anti - thyroid antibodies and the exclusion of other possible causes of encephalopathy , he was started on methylprednisolone 1 g / day for 3 days and was shifted over to prednisone 1 mg / kg / day . there was a marked improvement in the symptoms with no further episodes of myoclonus or seizures . one month after discharge , the patient reported a sustained improvement on all parameters , including memory and cognition , making it possible for his to return to an almost normal routine . prednisone dose was tapped over the period of 3 months without the recurrence of symptoms . hashimoto 's encephalopathy is a rare steroid responsive neuropsychiatric syndrome , associated with the serologic evidence of anti - thyroid antibodies , when other causes of encephalopathy are excluded . this syndrome was first described by brain et al . in 1966 , but it still remains a rare or under diagnosed condition with only about 130 cases reported in the literature until date , predominantly in adult females . the difficulty in diagnosis is due to the varied presentations and subtlety of symptoms , which may be acute or chronic . the pathophysiology of hashimoto encephalopathy is still unclear and the proposed pathogenetic mechanisms include autoimmune cerebral vasculitis , toxic effects of thyroid - stimulating hormone on the central nervous system and neuronal reaction mediated by antibodies . the anti - thyroid antibodies does not appear to be pathogenic and are considered as markers of the disease . based on this observation , it has been proposed to rename hashimoto 's encephalopathy ( he ) as steroid - responsive encephalopathy associated with autoimmune thyroiditis . the clinical manifestations usually include acute to the subacute onset of confusion with alteration of consciousness . two - thirds of patients may experience focal or generalized tonic - clonic seizures and 12% may present with status epileptics . hyperreflexia and other pyramidal tract signs are found in 85% of patients and psychosis , visual hallucinations and paranoid delusions have been reported in 25 - 36% of patients . myoclonus is a less publicized but common manifestation of he , with 52% patients diagnosed to have he , having a history of myoclonus . the diagnosis of he should be considered in patients presenting with the characteristic neuropsychiatric manifestations excluding other causes of encephalopathy . they should have ( 1 ) presence of high levels of anti - thyroid antibodies in serum or csf ; ( 2 ) no alteration in the csf and/or imaging tests compatible with infectious , vascular , or neoplastic etiology ; and ( 3 ) a good response to immunosuppressive therapy . elevated csf protein is a common and the thyroid profile may be variable , with 23 - 35% of patients having subclinical hypothyroidism , 17 - 20% having hypothyroidism , 7% having hyperthyroidism and 18 - 45% being euthyroid . non - specific eeg abnormalities are seen in 90 - 98% of patients and brain mri may show abnormalities in 49% such as cerebral atrophy , focal cortical abnormality , diffuse subcortical abnormality and non - specific subcortical focal white matter abnormality . the disease is responsive to immunosuppressive therapy and intravenous ( iv ) methylprednisolone ( 500 - 1000 mg / day ) for 3 - 5 days , followed by an oral dose of prednisone ( 1 - 2 mg / kg / day ) , followed by gradual tapering based on the clinical response being the commonly followed protocol . around 2 - 5% of patients may not respond to steroids , in whom azathioprine , iv immunoglobulins or plasmapheresis can be used with good results . the diagnosis of he should be considered in patients presenting with the characteristic neuropsychiatric manifestations excluding other causes of encephalopathy . they should have ( 1 ) presence of high levels of anti - thyroid antibodies in serum or csf ; ( 2 ) no alteration in the csf and/or imaging tests compatible with infectious , vascular , or neoplastic etiology ; and ( 3 ) a good response to immunosuppressive therapy . elevated csf protein is a common and the thyroid profile may be variable , with 23 - 35% of patients having subclinical hypothyroidism , 17 - 20% having hypothyroidism , 7% having hyperthyroidism and 18 - 45% being euthyroid . non - specific eeg abnormalities are seen in 90 - 98% of patients and brain mri may show abnormalities in 49% such as cerebral atrophy , focal cortical abnormality , diffuse subcortical abnormality and non - specific subcortical focal white matter abnormality . the disease is responsive to immunosuppressive therapy and intravenous ( iv ) methylprednisolone ( 500 - 1000 mg / day ) for 3 - 5 days , followed by an oral dose of prednisone ( 1 - 2 mg / kg / day ) , followed by gradual tapering based on the clinical response being the commonly followed protocol . around 2 - 5% of patients may not respond to steroids , in whom azathioprine , iv immunoglobulins or plasmapheresis can be used with good results .
blue rubber bleb nevus syndrome ( brbns ) also called bean 's syndrome is a rare disorder characterized by multiple cutaneous venous malformations in association with visceral lesions , most commonly affecting the gastrointestinal tract . we report here , a 21-year - old woman patient , who presented with unilateral , blaschkoid distribution of cutaneous venous malformations along with blue rubber bleb nevus and recurrent episodes of hematochezia due to vascular lesions in the sigmoid colon ; likely to be a case of brbns . the unusual unilateral , blaschkoid distribution of brbns prompted this present report .	blue rubber bleb nevus syndrome ( brbns ) is an uncommon condition with multiple venous malformations of the skin , gastrointestinal tract , and other visceral sites including the liver , spleen , bladder , spinal cord , lungs , and bones . clinically , the lesions are characteristically blue / purple soft compressible nodules with a rubbery feel . they are dome shaped , almost nipple - like rubber blebs , which may coalesce to form large masses . the gastrointestinal tract is the most common visceral site affected , and lesions share the same pathology as the cutaneous lesions . they are sessile or polypoid and affect all levels of the gastrointestinal tract , in particular , the small intestine . they are prone to bleeding and may cause a significant anemia requiring transfusion and iron replacement therapy . a 21-year - old lady presented to our outpatient department with multiple nontender , soft , rubbery , and compressible swellings distributed over the right side of the chest and right upper limb in a blaschkoid distribution . these lesions first appeared on the right side of the chest during her childhood and they gradually increased in number and size with age . her medical and family history were unremarkable except for the presence of recurrent episodes of painless bleeding per rectum ( hematochezia ) . cutaneous examination revealed multiple blue to purple papulonodules of variable sizes , with diameters ranging from 2 to 15 mm , distributed along right side of chest , the right hand and forearm [ figures 13 ] ; which on palpation yielded a feeling of cutaneous herniation . the affected limb was swollen , which when palpated yielded a bag - of - worm like feeling . clinically , the swellings were diagnosed to be blue rubber bleb nevi [ figures 4 and 5 ] , which were present in the background of cutaneous vascular malformation . bluish papulo - nodule over the right breast multiple skin colored swellings affecting the right hand multiple blue to purple cutaneous swellings affecting the right arm blue rubber bleb nevus on the background of swollen right arm photograph depicting the confinement of the lesions to the upper extremity of right side the hematological evaluation revealed iron deficiency anemia . doppler ultrasound hinted the presence of a low - flow lesion , which was suggestive of vascular malformation . computed tomography angiography of right upper extremity and upper chest revealed extensive venous angiomatous malformation in right superior extremity and right axilla , right side of upper chest wall , and right side of the neck with direct filling of contrast from upper part of superior vena cava . the malformation involved subcutaneous fat planes , muscles , and intermuscular portions of the right upper chest wall , right axilla , and right superior extremity [ figures 6 and 7 ] . colonoscopy detected the presence of vascular polyps in her sigmoid colon [ figure 8 ] , which presumably led to the recurrent episodes of hematochezia . currently , the patient has been put under oral iron supplements and has been referred to the department of plastic surgery . computed tomography angiogram of showing extensive vascular malformation affecting the right upper limb along with right - upper trunk . ct angiogram showing extensive vascular malformation affecting the right upper limb along with right - upper trunk . blue rubber bleb nevus syndrome is an uncommon condition characterized by multiple venous malformations affecting the skin and internal viscera , gastrointestinal tract involvement being the most common . it was first described by gascoyen in 1806 , but in 1958 , william bennet bean coined the term blue rubber nevus syndrome for its color and consistency . brbns affects both sexes and is usually sporadic , although autosomal dominant inheritance has been reported . bean described three types of lesions in brbns , which are often noted at birth or during the neonatal period , although they can present later . the first type is the compressible , red - blue , nipple - like lesions . the second type is blue - black nonblanching macular lesions and the third type is subcutaneous , vascular , soft tissue lesions . in our case , the patient presented with lesions akin to the first type . in the english literature , most of the lesions are diffusely distributed all over the body , with blue subcutaneous nodules as the characteristic lesions . however , in our case , the skin lesions are distributed unilaterally , affecting only the right side of the body . the lesions on the upper limb are also arranged in a linear fashion making the case even rarer . to the best of our knowledge , there is only a single report of brbns with linear , unilateral distribution in the world literature , and this happens to be the second case showing such a unique distribution . complications include acute hemorrhage , iron deficiency anemia following chronic bleeding , thrombocytopenia , disseminated intravascular coagulation , intussusception , volvulus , bowel infarction , skeletal bowing , pathologic fracture , bony overgrowth , articular derangement and pain due to phleboliths . the presence of cutaneous lesions should alert clinicians on the possible presence of internal vascular malformations . in our present case , the recurrent episodes of hematochezia possibly occurred due to underlying vascular malformations ( polyps ) present in her sigmoid colon , which were detected on colonoscopy . these include cryotherapy , sclerotherapy , surgery , and newly - reported long - pulsed neodymium : yttrium - aluminium - garnet 1064 nm laser for cutaneous lesions . we have referred our patient to the department of plastic surgery for possible surgery . while the coexistence of cutaneous and gastrointestinal venous malformations are the hallmark of brbns , venous malformations in additional sites , such as the oral cavity , central nervous system , endocrine glands , musculoskeletal system , kidney , bladder , liver , spleen , heart , and lungs , the extra - cutaneous and gastrointestinal lesions may present with hemoptysis , hematuria and skeletal deformities or be asymptomatic . the differential diagnoses include maffucci syndrome , venous lakes , osler weber rendu syndrome or hereditary hemorrhagic telangiectasia , disseminated hemangiomatosis and kaposi sarcoma . brb nevi can be differentiated from the above conditions based on the latter 's characteristic features such as bluish , bleb - like , painful , tender , hyperhidrotic , compressible nodules with feeling of dermal herniation and associated gastrointestinal tract involvement . a complete history and physical evaluation are a must to make a successful diagnosis to help in treatment planning and to prevent fatal potential complications . linear , unilateral distribution of brbns is extremely uncommon ; this happens to be the second such case in the english literature to the best of our knowledge . unilateral , linear or blaschkoid distribution of blue rubber bleb nevus syndrome is extremely uncommon ; this happens to be the first such case from india .
antimalarial bioassay - guided fractionation of an etoh extract of the root wood of cryptocarya rigidifolia ( lauraceae ) led to the isolation of the five new 5,6-dihydro--pyrones cryptorigidifoliols a e ( 15 ) and the six bicyclic tetrahydro--pyrone derivatives cryptorigidifoliols f k ( 611 ) . the structure elucidations of all compounds were made on the basis of the interpretation of spectroscopic data and chemical derivatization , and the relative and absolute configurations were determined by noesy , electronic circular dichroism ( ecd ) , and 1h nmr analysis of -methoxyphenylacetyl ( mpa ) derivatives . the bicyclic tetrahydro--pyrone derivatives were identified as products of acid - catalyzed intramolecular michael addition of the 5,6-dihydro--pyrones in the presence of silica gel . a structure activity relationship study suggested that the presence of an ,-unsaturated carbonyl moiety is not essential for potent antimalarial activity .	the etoac - soluble fraction obtained from a liquid liquid partition of the etoh extract ( 100 mg ) showed antiplasmodial activity . dereplication as previously described indicated that the extract contained at least one new bioactive compound , so a larger sample was investigated . fractionation of the etoac - soluble fraction of this sample by chromatography on sephadex lh-20 , reverse - phase spe , normal - phase silic - gel column chromatography , and c18 hplc yielded compounds 1 and 68 , together with fractions that were mixtures of 5,6-dihydro--pyrones and bicyclic tetrahydro--pyrones . purification of these fractions was effected by diol ptlc or hplc to yield compounds 2 , 3 , 5 , and 911 . compound 1 was isolated as a clear oil , and its molecular formula was established as c20h36o3 by hresims ( m / z 325.2753 [ m + h ] ) . its ir spectrum showed absorptions at 3340 , 1719 , and 1613 cm assigned to a hydroxy group and an ,-unsaturated -lactone moiety , respectively . its h nmr spectroscopic data had signals for a conjugated olefinic moiety at h 6.90 ( m , 1h , h4 ) and 6.03 ( dt , j = 9.8 , 1.7 hz , 1h , h3 ) , two oxymethine groups at h 4.75 ( m , 1h , h6 ) and 4.00 ( brs , 1h , h2 ) , a terminal primary methyl group ( h 0.88 , t , j = 7.0 hz , 3h , ch315 ) in the upfield region , and a multiplet at h 2.36 ( 2h , h5 ) , representing a de - shielded methylene group . in the hmbc spectrum , the methylene protons at h 2.36 ( h5 ) showed correlations both to an olefin group ( c 121.3 , c3 , and 145.0 , c4 ) and to the oxymethine resonance at h 4.75 and c 74.8 ( c6 ) . in addition , both the olefinic protons ( h 6.90 , h4 , and h 6.03 , h3 ) and the oxymethine at h 4.75 ( h6 ) correlated with a carbonyl carbon at c 164.6 ( c2 ) ( figure 1 ) . these data indicated a six - substituted 5,6-dihydro--pyrone , a common ring system of secondary metabolites found in cryptocarya species . the remaining oxymethine group at h 4.00 ( 1h , h2 ) was assigned to c2 , which was flanked by two methylenes ( c1 and c3 ) as indicated by hmbc correlations ( figure 1 ) from the methylene protons at h 1.92 ( ddd , j = 14.5 , 9.6 , 2.2 hz , h1 ) and h 1.65 ( m , h1 ) to c5 ( c 30.1 ) , from h2 ( h 4.00 ) to the oxymethine carbon at c 74.8 ( c6 ) , and from h2 to the two neighboring methylene carbons at c 41.2 ( c1 ) and c 38.2 ( c3 ) . the 6r absolute configuration was determined by the positive cotton effects at 256 nm observed in its ecd spectrum in meoh , arising from the n * transitions of the lactone ring . the h nmr spectra of the ( r ) and ( s ) mpa ester derivatives of 1(26 ) revealed slight differences in the h chemical shifts of c6 and adjacent protons that allowed the assignment of the r configuration to c2 of 1 ( figure s1a ) . the complete assignment of all protons and carbons of 1 ( table 1 ) was accomplished by further interpretation of its hmbc and hsqc spectra . compound 1 was thus assigned as 6r-(2r - hydroxypentadecyl)-5,6-dihydro-2h - pyran-2-one and named cryptorigidifoliol a. spectra obtained in cdcl3 ; assignments on the basis of analysis of 2d nmr spectra . data ( ) measured at 500 mhz ; brs = broad singlet , brd = broad doublet , t = triplet , ddd = doublet of doublets of doublets , dt = doublet of triplets , m = multiplet . j values are in hz and are omitted if the signals overlapped as multiplets . data ( ) measured at 125 mhz ; ch3 , ch2 , ch , and c multiplicities were determined by an hsqc experiment . compound 2 was obtained as an oil with the molecular formula c24h44o4 on the basis of its hresims spectrum ( m / z 397.3317 [ m + h ] ) . the uv , ir , and h nmr spectroscopic data of 2 were comparable to those of 1 , suggesting that 2 is also a six - substituted 5,6-dihydro--pyrone . the major difference between the h nmr spectroscopic data of 1 and that of 2 was the presence of an additional signal for an oxymethine proton at h 3.89 ( m , 1h , h4 ) in 2 . the hmbc correlation between the oxymethine proton and the carbon signal at c 69.8 ( c2 ) assigned the additional hydroxy group to c4 , and this assignment is supported by the hmbc cross peaks between the two adjacent methylene protons ( h 1.61 , m , 2h , h3 ; 1.47 , m , 2h , h5 ) and the oxymethine carbon at c 74.0 . similar to 1 , the complete assignments of all protons and carbons of 2 ( table 1 ) were accomplished by interpretation of its hmbc and hsqc spectra . ecd and synthesis and h nmr analysis of its mpa esters were used to assign the configurations of c6 , c2 , and c4 as r , s , and r , respectively . compound 2 was thus assigned as 6r-(2s,4r - dihydroxynonadecyl)-5,6-dihydro-2h - pyran-2-one and has been named cryptorigidifoliol b. the molecular formula of compound 3 ( c22h38o3 , hresims m / z 351.2902 [ m + h ] ) and its h nmr spectrum ( h 5.34 , m , 2h , h10 and h11 ) indicated that it had an additional disubstituted olefin moiety as compared with compound 1 . its uv , ir , and h nmr spectroscopic data indicated the presence of the same -pyrone ring as 1 and 2 , and hmbc correlations from h10 and h11 ( h 5.34 ) to the carbons at c 27.0 indicated that the additional olefinic moiety must be located between two methylene groups . long - range correlation from both h9 and h12 ( h 2.051.99 , m , 4h ) and h8 and 13 ( h 1.371.31 , m , 4h ) to the carbons at c10 and 11 ( c 130 ) in the hmbc spectrum assigned the olefinic moiety to c10 and c11. the geometry of the double bond was assigned as z on the basis of the shielded c nmr chemical shift of the methylenes connected to the double bond ( c 29.4 ) . the position of the double bond within the alkyl chain was determined unambiguously by analysis of the gc - eims fragmentation of the dimethyldisulfide ( dmds ) derivative of 3 , which showed a major ion at m / z 299 attributable to fragmentation between the two ch3s groups located at the original site of unsaturation . fragment ions at m / z 281 and 145 were also observed and support the assigned structure ( figure s2a ) . the relative configuration of 3 and the assignment of its h and c nmr data were determined by the same methods as for 1 and 2 . compound 3 was assigned as 6r-(2r - hydroxy-10z - heptadecenyl)-5,6-dihydro-2h - pyran-2-one and is named cryptorigidifoliol c. the molecular formula of 4 ( c24h42o3 , hresims m / z 396.3489 [ m + nh4 ] ) differed from the molecular formula of 3 ( c22h38o3 ) only by a c2h4 unit . inspection of the h nmr spectra of 3 and 4 demonstrated that 4 possessed a structure similar to that of 3 but with two additional carbons in the alkenyl chain . the intense fragment ions at m / z 299 and 173 and the additional ion peak at m / z 281 in the eims spectrum of the dmds adduct of 5 indicated that the position of the double bond was between c10 and c11 ( figure s2b ) . the complete nmr data and configurations of all stereogenic centers in 4 were assigned by the same methods as for 13 . compound 4 was assigned as 6r-(2s - hydroxy-10z - nonadecenyl)-5,6-dihydro-2h - pyran-2-one and is named cryptorigidifoliol d. the h nmr spectrum of compound 5 ( c24h42o4 , hresims m / z 377.3060 [ m oh ] ) showed the presence of two oxymethine groups ( h 4.63 , m , 1h , h2 ; h 4.15 , m , 1h , h12 ) and a double bond ( h 5.68 , m , 1h , h4 ; h 5.49 , dd , j = 15.3 , 7.0 , 1h , h3 ) in the alkyl chain , besides the -pyrone signals at h 6.90 , 6.03 , 4.69 , and 2.44 . the large coupling constant ( 15.3 hz ) observed for h3 indicated the e geometry of the double bond . in the hmbc spectrum , correlations were observed between the protons at h 1.79 and 1.73 ( each m , 1h , h1 ) and c3 ( c 131.4 ) and between the proton at h 4.63 ( m , 1h , h2 ) and c4 ( c 132.6 ) . these observations suggested the connection of the olefinic moiety with the c2 oxymethine functionality . the eims data of 5 showed significant ions at m / z 265 and 247 , together with a less intense ion at m / z 111 , consistent with assignment of the second hydroxy group to c12 ( figure s3 ) . the configurations at c6 and c2 were assigned to be r and s , respectively , by interpretation of the ecd spectroscopic data and by the mpa ester method . an attempt was made to determine the configuration at c12 by using the mpa ester method , but it did not lead to any firm conclusion because we could not distinguish the chemical shifts of the protons of the two methylene groups attached to c12. compound 5 was assigned as 6r-(2r,12-dihydroxy-3e - nonadecenyl)-5,6-dihydro-2h - pyran-2-one and named cryptorigidifoliol e. compound 6 had the molecular formula of c24h42o4 on the basis of its hresims ( m / z 395.3149 [ m + h ] ) . its ir spectrum showed the absorptions characteristic of a -lactone moiety ( 1719 and 1073 cm ) . the h nmr spectrum lacked the signals for the vinylic protons of an ,-unsaturated lactone unit , and the ir absorption at 1615 cm found in compounds 15 was absent . a new methylene signal was observed at h 2.89 ( brd , j = 19.3 hz , 1h , h4a ) and 2.78 ( dd , j = 19.3 , 4.5 hz , 1h , h4b ) , which showed hmbc correlations with a carbonyl carbon at c 169.7 ( c3 , figure 2 ) . a signal for a vinylic proton was observed in the h nmr spectrum at h 5.33 ( m , 2h , h8 and h9 ) , and the four indices of hydrogen deficiency of 6 thus required a second ring in addition to the lactone and the double bond functionalities . the h nmr spectrum showed the presence of four oxymethine protons ( h 4.89 ( m , 1h , h1 ) , 4.36 ( t , j = 4.5 hz , 1h , h5 ) , 4.10 ( m , 1h , h7 ) , and 3.81 ( m , 1h , h2 ) ) directly attached to c1 ( c 73.1 ) , c5 ( c 66.0 ) , c6 ( c 63.6 ) , and c2 ( c 68.3 ) , respectively , assignments that were confirmed by hsqc data . in the hmbc spectrum , cross peaks were observed from the two oxymethine protons at h 4.89 ( h1 ) and 4.36 ( h5 ) to the carbonyl carbon at c 169.7 ( c3 ) and the c7 oxymethine ( c 63.6 ) . correlations were also observed between the two oxymethine protons h1 and h5 and c5 ( c 66.0 ) and c1 ( c 73.1 ) , respectively , and between the oxymethine proton at h 4.10 ( h7 ) and the oxygenated carbon ( c 68.3 ( c2 ) ; h 3.81 ( m , 1h , h2 ) ; figure 2 ) . collectively , these correlations permitted assignment of the bicyclic ring system , the alkyl chain substituted at c7 , and a hydroxy group at c2 of 6 ( figure 2 ) . the long - range cross peaks observed between h8 and h9 ( h 5.33 , m , 2h ) and c 29.6 in the hmbc spectrum indicated that the allylic methylene groups resonated at c 29.6 ( c7 and c10 ) and indicated the z geometry of the sole double bond in the alkyl chain . the position of this double bond was assigned to c8/9 on the basis of the eims fragmentation of the dmds adduct , which had an intense peak at m / z 173 and a peak at m / z 297 corresponding to loss of water from the lactone - containing fragment ion ( figure s2c ) . an attempt to assign the configuration at c2 of 6 by the mpa ester method was not successful because there were no significant ( h ) differences between its r- and s - mpa derivatives . because bicyclic tetrahydro--pyrones such as 6 are formed from the corresponding 5,6-dihydro--pyrones , as explained below , and all of these 5,6-dihydro--pyrones have the 6r configuration , it is proposed that the orientation of substituents at c1 of 6 ( corresponding to c6 in the putative monocyclic precursor ) must be the same . because of a change in group priorities , the c1 absolute configuration is s. the preference for the formation of a less - strained cis fused bicyclic system dictates the generation of a 5r - configured center . the same cis fused configuration was demonstrated for some related bicyclic tetrahydro--pyrones by x - ray crystallography . the configuration of 6 at c7 could not be assigned because its 5,6-dihydro--pyrone precursor was not isolated . the complete nmr assignments of 6 ( table 2 ) were facilitated by further interpretation of its hmbc and hsqc spectra . compound 6 was thus assigned as ( 1s,5r)-7-(2-hydroxy-8z - heptadecenyl)-2,6-dioxabicyclo[3.3.1]nonan-3-one and has been named cryptorigidifoliol f. spectra obtained in cdcl3 ; assignments are on the basis of analysis of 2d nmr spectra . data ( ) measured at 500 mhz ; s = singlet , br d = broad doublet , dd = doublet of doublets , ddd = doublet of doublets of doublets , m = multiplet . j values are in hz and are omitted if the signals overlapped as multiplets . data ( ) measured at 125 mhz ; ch3 , ch2 , ch , and c multiplicities were determined by an hsqc experiment . compounds 7 ( m / z 421.3662 [ m + h ] ) and 8 ( m / z 355.2831 [ m + h ] ) also contained a bicyclic tetrahydro--pyrone ring . comparison of their molecular weights and h nmr spectra with those of cryptorigidifoliol f ( 6 ) revealed that the only structural differences were in the alkyl chain length and the absence or presence of a double bond or hydroxy group on the alkyl chain . cryptorigidifoliol g ( 7 ) , with the molecular formula c27h48o3 , was a bicyclic tetrahydro--pyrone similar to 6 but lacked the c2 hydroxy group and had a 20-carbon alkenyl chain at c7 . preparation of its dmds derivative permitted assignment of the double bond at c8 ( m / z at 299 and 215 ; figure s2c ) . its configuration at c7 could not be determined because its presumed monocyclic precursor was not isolated . cryptorigidifoliol h ( 8) was also a bicyclic tetrahydro--pyrone similar to 6 but with a saturated alkyl chain at c7 ; the chain length was determined to be 14c on the basis of its molecular formula of c21h38o4 . as in the case of 7 , its configuration at c7 could not be determined . compounds 911 ( cryptorigidifoliols i k ) are the bicyclic derivatives of 2 , 3 , and 5 , as determined by the conversions described below and by comparison of their h nmr and hresims data with those of their 5,6-dihydro--pyrone precursors and those of compounds 68 . because the configurations of the precursors 2 , 3 , and 5 at the 2 position have been established , the corresponding configurations at c7 of 9 , 10 , and 11 were assigned as s , r , and r , respectively . compounds 6 , 8 , 9 , and 11 had no significant ( h ) difference between their r- and s - mpa derivatives , thus precluding assignment of absolute configurations at these centers . the initial purification of the bioactive etoac fraction involved sephadex lh-20 column chromatography , reverse - phase spe , normal - phase silica - gel column chromatography , and c18 hplc . this procedure yielded fractions that in most cases were mixtures of 5,6-dihydro--pyrones and bicyclic tetrahydropyrones . in addition , it was noted that the early - eluting fractions from the open silica - gel column contained either pure 5,6-dihydro--pyrone 1 or mixtures of 5,6-dihydro--pyrones 2 , 3 , and 5 and bicyclic tetrahydropyrones 911 . the later - eluting fractions , however , yielded only the bicyclic tetrahydropyrones 68 . this suggested that the 5,6-dihydro--pyrones were cyclized during their exposure to silica gel , with the more polar later - eluting compounds that were exposed for a longer time to silica gel becoming completely converted to cyclized product . pure 5,6-dihydro--pyrone 2 and related compounds were thus prepared by purification by ptlc on diol silica gel , which did not induce intramolecular cyclization . it is worth noting that grkovic and co - workers isolated only 5,6-dihydro--pyrones , without artifacts , by using diol column chromatography during their bioassay - guided fractionation of a papua new guinea species of cryptocarya in order to find compounds that can rescue pdcd4 from tpa - induced degradation . to verify the cyclization hypothesis , silica gel was mixed with compound 2 in meoh / hexanes / etoac and the resulting suspension was allowed to stand for 3 h at room temperature . examination of the resulting solution showed that only compound 9 was present , confirming that silica gel catalyzed the cyclization of the 5,6-dihydro--pyrone 2 to the bicyclic tetrahydropyrone 9 ( scheme 1 ) . compounds 3 and 5 were also treated in the same way , leading to the formation of the corresponding bicyclic tetrahydropyrone derivatives 10 and 11 . these observations supported the hypothesis that the bicyclic tetrahydropyrones are formed by intramolecular cyclization of c2-hydroxylated 5,6-dihydro--pyrones . however , this does not rule out the possibility that at least some bicyclic tetrahydropyrones are formed in the plant , and examination of the crude extract by h nmr spectroscopy indicated that it did contain signals consistent with the presence of bicyclic tetrahydropyrones . this result could be due to either the presence of the cyclized compounds in the plant or intramolecular cyclization during the extraction of the plant and the processing of the extract in madagascar prior to analysis . regrettably , we did not have access to fresh plant material to test these alternate hypotheses , but the fact that chromatography over silica gel was used in every case where the cyclized compounds are reported strongly suggests that these compounds are indeed formed during the purification process . compounds 111 were evaluated for their antiparasitic activity against the chloroquine / mefloquine - resistant dd2 strain of plasmodium falciparum . compounds 17 exhibited moderate antimalarial activity , with ic50 values of 9.2 0.9 , 5.8 1.4 , 5.5 0.7 , 9.0 3.0 , 4.0 2 , 7.4 0.6 , and 6.0 0.5 m , respectively . compounds 811 were all less active , with ic50 values > 10 m ( tables 3 and 4 ) . these data indicate that the presence of the ,-unsaturated carbonyl moiety is not essential for antimalarial activity because bicyclic compounds 6 and 7 have activity comparable to that of ,-unsaturated carbonyl compounds 15 . antimalarial activity against the dd2 strain of plasmodium falciparum antiproliferative activity against human ovarian cancer cells . antimalarial activity against the dd2 strain of plasmodium falciparum antiproliferative activity against human ovarian cancer cells . all of the compounds were also evaluated for their antiproliferative activity against a2780 human ovarian cancer cells , but only compound 3 had an ic50 value less than 10 m ( tables 3 and 4 ) . because these compounds have only moderate antiparasitic activities , significant improvement in their potency and therapeutic index will be necessary before they can be used as lead compounds for the development of new antiplasmodial drugs . the formation of compounds 611 serves to highlight the fact that 5,6-dihydro--pyrones containing c2 hydroxy groups in the side chain are susceptible to cyclization in the presence of silica gel at room temperature . it is thus possible that the bicyclic tetrahydropyrones previously reported were also formed by cyclization of the corresponding 5,6-dihydro--pyrones during the isolation process . this finding also provides support for the general belief that silica gel should be avoided for the isolation of most natural products because it will not in general be known whether or not the unknown compounds might be susceptible to similar unwanted reactions . in place of silica gel , diol and c18 ir and uv spectra were measured on an midac m - series ftir and shimadzu uv-1201 spectrophotometers , respectively . 1d and 2d nmr spectra were recorded on a bruker avance 500 spectrometer in cdcl3 or pyridine - d5 ( with cdcl3 or pyridine - d5 as reference ) . high - resolution esi mass spectra were obtained on an agilent 6220 mass spectrometer . open - column chromatography was performed using sephadex lh-20 and silica gel ( 230 - 400 mesh , silicycle co. , usa ) . semipreparative hplc was performed using shimadzu lc-10at pumps coupled with a semipreparative phenomenex c18 column ( 5 m , 250 10 mm ) and semipreparative varian diol column ( 250 10.0 mm ) , a shimadzu spd m10a diode array detector , and a scl-10a system controller . preparative hplc was performed using shimadzu lc-8a pumps coupled with a preparative varian phenomenex c18 column ( 250 21.4 mm ) , a shimadzu spd m10a diode array detector , and an scl-10a system controller . preparative tlc was performed using diol plates ( 500 m / g , sorbtec co. , usa ) . all isolated compounds were purified to 95% purity or better , as judged by hplc ( both uv and elsd detection ) before determining bioactivity . plant parts of cryptocarya rigidifolia van der werff ( lauraceae ) were collected and authenticated by f.r . and co - workers at an elevation of about 1000 m from a 13 m tall tree with a diameter of 13 cm at chest height . the tree had round green fruit , with the remains of brown calyx above . collection was made near the towns of imerimandroso and antanandava in the district of ambatondrazaka , north of the zahamena national park , at coordinates 172845 s , 484410 e. the tree was growing in medium - altitude rainforest on the edge of an old area of slash - and - burn agriculture . duplicate voucher specimens ( ratovoson 250 ) were deposited at the centre national dapplication des recherches pharmaceutiques ( cnarp ) , the herbarium of the department of forestry and fishery research ( tef ) , and the missouri botanical garden , st . a ground sample of c. rigidifolia root wood ( 310 g ) was extracted with etoh ( 1000 ml ) at room temperature to yield 16.3 g of crude etoh extract designated mg 0441 , and a portion of this extract was made available to virginia tech for bioassay - guided isolation . the crude etoh extract ( 5.0 g ) was dissolved in 90% aqueous meoh ( 60 ml ) and extracted with hexanes . the 90% aqueous meoh layer was evaporated , suspended in h2o ( 100 ml ) , and extracted with etoac ( 100 ml ) . the antimalarial activities were concentrated in the etoac ( ic50 = 2.55 g / ml ) and hexanesfractions ( ic50 5 g / ml ) . the etoac fraction ( 971 mg ) was subjected to sephadex lh-20 size chromatography ( ch2cl2/meoh = 1:1 ) to give four fractions , and the most active fraction fr-2 ( ic50 = 2.55 g / ml ) was fractionated by c18 spe using 70% aqueous meoh , 100% meoh , and ch2cl2 separately . the 100% meoh fraction ( ic50 = 1.252.5 g / ml ) was divided into eight subfractions by silica - gel column chromatography ( hexanes / etoac from 1:1 to 3:7 to 1:9 , and a final elution with meoh ) . the third subfraction ( ic50 < 1.25 g / ml ) was subjected to c18 hplc with a solvent gradient of h2o / ch3cn , 20:80 to 10:90 from 0.01 to10 min , to 0:100 from 10 to 15 min , and ending with 100% ch3cn to 30 min , furnishing compound 1 ( tr = 20.2 min , 1.1 mg , ic50 = 2.98 g / ml ) and an active fraction ( tr = 20.8 min ) . further purification of the active fraction by diol ptlc ( hexanes / etoac = 4:6 ) yielded 3 ( 1.4 mg , ic50 = 1.93 g / ml ) and 10 ( 0.4 mg , ic50 = 5.74 g / ml ) . the fourth subfraction from silica - gel column chromatography was subjected to c18 hplc with a solvent gradient of h2o / ch3cn , 30:7020:80 from 0.01 to15 min , to 10:90 from 15 to 35 min , to 0:100 from 35 to 50 min , and ending with 100% ch3cn to 60 min . purification by diol ptlc ( hexanes / etoac = 4:6 ) of the mixtures with tr = 37.5 and 40.2 min obtained from this process gave compounds 5 ( 0.9 mg , ic50 = 3.55 g / ml ) , 11 ( 3.1 mg , ic50 > 20 g / ml ) , 2 ( 1.3 mg , ic50 = 2.30 g / ml ) and 9 ( 0.5 mg , ic50 = 6.57 g / ml ) . the fifth subfraction from silica - gel column chromatography was further purified by c18 hplc by using the same profile as the fourth subfraction , giving pure compounds 8 ( 4.2 mg , tr = 38 min , ic50 = 13.6 g / ml ) and 6 ( 9.6 mg , tr = 40.8 min , ic50 = 1.58 g / ml ) . the hexanes fraction was subjected to sephadex lh-20 gel chromatography ( ch2cl2/meoh = 1:1 ) to give three fractions , and the most active fraction fr-2 ( ic50 2.55 g / ml ) was fractionated by preparative c18 hplc with a solvent gradient of h2o / ch3cn , 30:700:100 from 0.01 to 30 min , and ending with 100% ch3cn to 45 min . this process yielded compound 7 ( 1.6 mg , tr = 39 min , ic50 = 2.50 g / ml ) and an active fraction ( tr = 32.5 min ) . further purification of the fraction by diol hplc with a solvent gradient of hexanes / etoac , 90:1080:20 from 0.01 to 10 min , to 70:30 from 10 to 15 min , to 60:40 from 15 to 20 min , to 0:100 from 20 to 25 min , and ending with 100% etoac wash to 35 min , afforded compound 4 ( 0.9 mg , ic50 2.76 g / ml ) . colorless oil ; [ ]d + 6 ( c 0.5 , meoh ) ; uv ( meoh ) max ( log ) 204 ( 3.63 ) nm ; ir max 3340 , 2922 , 2849 , 1719 , 1613 , 1089 cm ; ecd ( c 0.031 mm , meoh ) max ( ) 203 ( + 8.5 ) , 235 ( + 2.4 ) , 256 ( + 3.8 ) ; h and c nmr data , see table 1 ; hresims m / z [ m + h ] 325.2753 ( calcd for c20h37o3 , 325.2737 ) . colorless oil ; [ ]d + 9 ( c 0.1 , meoh ) ; uv ( meoh ) max ( log ) 204 ( 3.83 ) nm ; ir max 3343 , 2922 , 2850 , 1714 , 1615 , 1092 cm ; ecd ( c 0.031 mm , meoh ) max ( ) 209 ( + 9.7 ) , 235 ( + 1.9 ) , 256 ( + 3.5 ) ; h and c nmr data , see table 1 ; hresims m / z [ m + h ] 397.3317 ( calcd for c24h45o4 , 397.3312 ) . colorless oil ; [ ]d + 19 ( c 0.6 , meoh ) ; uv ( meoh ) max ( log ) 204 ( 3.91 ) nm ; ir max 3439 , 2924 , 2855 , 1728 , 1623 , 1044 cm ; ecd ( c 0.031 mm , meoh ) max ( ) 207 ( + 9.3 ) , 235 ( + 1.2 ) , 255 ( + 3.1 ) ; h and c nmr data , see table 1 ; hresims m / z [ m + h ] 351.2902 ( calcd for c22h39o3 , 351.2894 ) . colorless oil ; [ ]d + 18 ( c 0.6 , meoh ) ; uv ( meoh ) max ( log ) 204 ( 3.50 ) nm ; ir max 3444 , 2922 , 2855 , 1724 , 1625 , 1034 cm ; ecd ( c 0.031 mm , meoh ) max ( ) 208 ( + 10.6 ) , 235 ( + 1.6 ) , 256 ( + 3.0 ) ; h and c nmr data , see table 1 ; hresims m / z [ m + nh4 ] 396.3489 ( calcd for c24h46no3 , 396.3472 ) . colorless oil ; [ ]d 25 ( c 0.4 , meoh ) ; uv ( meoh ) max ( log ) 204 ( 3.24 ) nm ; ir max 3382 , 2922 , 2845 , 1729 , 1625 , 1077 cm ; ecd ( c 0.031 mm , meoh ) max ( ) 207 ( + 11.0 ) , 234 ( + 2.3 ) , 256 ( + 3.2 ) ; h and c nmr data , see table 1 ; hresims m / z [ m colorless oil ; [ ]d 10 ( c 0.5 , meoh ) ; uv ( meoh ) max ( log ) 204 ( 3.14 ) nm ; ir max 3450 , 2932 , 2855 , 1719 , 1073 cm ; h and c nmr data , see table 2 ; hresims m / z [ m + h ] 395.3149 ( calcd for c24h43o4 , 396.3156 ) . colorless oil ; [ ]d 4 ( c 0.5 , meoh ) ; uv ( meoh ) max ( log ) 204 ( 3.14 ) nm ; ir max 3450 , 2932 , 2855 , 1719 , 1073 cm ; h and c nmr data , see table 2 ; hresims m / z [ m + h ] 421.3662 ( calcd for c27h49o3 , 421.3676 ) . colorless oil ; [ ]d 11 ( c 0.5 , meoh ) ; uv ( meoh ) max ( log ) 204 ( 2.66 ) nm ; ir max 3540 , 2917 , 2845 , 1719 , 1070 cm ; h and c nmr data , see table 2 ; hresims m / z [ m + h ] 355.2831 ( calcd for c21h39o4 , 355.2843 ) . colorless oil ; [ ]d 8 ( c 0.4 , meoh ) ; uv ( meoh ) max ( log ) 204 ( 3.43 ) nm ; ir max 3340 , 2943 , 2846 , 1727 , 1068 cm ; h and c nmr data , see table 2 ; hresims m / z [ m + h ] 397.3317 ( calcd for c24h45o4 , 397.3312 ) . colorless oil ; [ ]d + 4 ( c 0.6 , meoh ) ; uv ( meoh ) max ( log ) 204 ( 3.62 ) nm ; ir max 3345 , 2939 , 2844 , 1739 , 1056 cm ; h and c nmr data , see table 2 ; hresims m / z [ m + h]351.2902 ( calcd for c22h39o3 , 351.2894 ) . colorless oil ; [ ]d 8 ( c 0.6 , meoh ) ; uv ( meoh ) max ( log ) 204 ( 3.10 ) nm ; ir max 3431 , 2956 , 2867 , 1732 , 1036 cm ; h and c nmr data , see table 2 ; hresims m / z [ m + h ] 377.3060 ( calcd for c24h41o3 , 377.3050 ) . pure samples ( 200 g ) dissolved in 200 l of hexanes were treated with dimethyldisulfide ( 400 l ) and 12 drops of iodine solution ( 6% w / v in ether ) in a 6 dram vial at 40 c for 30 h. the mixture was cooled and diluted with hexanes ( 1 ml ) , and iodine was removed by extracting with thiosulfate ( 1 ml , 5% w / v ) . the organic layer was removed , and the water layer was re - extracted with hexanes / ch2cl2 ( 1:1 ) . the combined organic layers were dried and subjected to analysis by gc - eims . the in - nmr - tube reaction was carried out to prepare the mpa ester derivatives . the selected 5,6-dihydro--pyrone ( 0.2 mg ) was dissolved in pyridine - d5 in a clean nmr tube , and r-()--methoxyphenylacetyl ( mpa ) chloride ( 0.5 mg ) was added to the nmr tube under a n2 gas flow , and the nmr tube was gently shaken . the nmr tube was kept at room temperature overnight and was then analyzed by h nmr spectroscopy . the s - mpa esters of the 5,6-dihydro--pyrones were prepared from s - mpa chloride by the same method . compound 2 ( 0.2 mg ) was dissolved in a mixture of meoh ( 2 ml ) , etoac ( 0.5 ml ) , and hexanes ( 0.5 ml ) . enough silica - gel powder was added to the solution to absorb it , and the mixture was kept at room temperature for 3 h. the resulting damp powder was eluted with meoh , and the eluate concentrated on a rotavapor . compounds 3 and 5 ( each 0.2 mg ) were also treated separately and worked up under the same conditions . the products were identified as 10 and 11 , respectively , by h nmr spectroscopy . the a2780 ovarian cancer cell line antiproliferative bioassay was performed at virginia tech as previously reported . paclitaxel was used as the positive control ; it had an ic50 value of 73 15 nm . the effect of each compound on parasite growth of the dd2 strain of p. falciparum was measured in a 72 h treatment using the malaria sybr green i - based fluorescence assay as described previously . artemisinin was used as the positive control ; it had an ic50 value of 7 1 nm .
liver cancer , primarily hepatocellular carcinoma ( hcc ) , is a major cause of cancer - related death worldwide . hcc is a suitable model of inflammation - induced cancer because more than 90% of hcc cases are caused by liver damage and chronic inflammation . several inflammatory response pathways , such as nf-b and jak / stat3 signaling pathways , play roles in the crosstalk between inflammation and hcc . micrornas ( mirnas ) are evolutionarily conserved , short endogenous , non - coding single - stranded rnas that are involved in various biological and pathological processes by regulating gene expression and protein translation . evidence showed that mirnas play a pivotal role in hepatitis virus infection and serve as promoters or inhibitors of inflammatory response . aberrant mirna was observed during liver inflammation and hcc . many dysregulated mirnas modulate the initiation and progression of inflammation - induced hcc . this review summarizes the role and functions of mirnas in inflammation - associated hcc , as well as the designed therapeutics targeting mirnas to treat liver inflammation and hcc .	the implication of inflammation in the development of cancers is well recognized . in the 19th century , virchow observed leukocytes in tissues that contained inflammatory infiltrates , which later on was confirmed in several clinical cases . however , these insights were not further examined until data collected from epidemiological studies and experimental models showed a link between chronic inflammation and various types of cancers . chronic inflammation occurs with continuous inflammatory signaling caused by viral , bacterial , and parasitic infections or chemical irritants ; exposure to chemical irritants results in recruitment of inflammatory cells , increased production of reactive oxygen species ( ros ) , and reduced dna repair . most cancer tissues show specific types of inflammatory response ; this subset accounts for approximately 20% of human cancers worldwide and is considered an essential component of tumorigenesis , including tumor initiation , progression , growth , and metastasis . liver cancer is one of the most difficult cancers to treat and is the second leading cause of cancer death worldwide , especially in china , where it accounts for half of total cancer incidence and death . liver cancer is strongly correlated with viral hepatitis infection ; more than 90% of liver cancers are associated with chronic hepatitis or liver cirrhosis . however , cell death - regeneration cycles are driven by persistent inflammation that occurs when inflammation - associated signaling can not be inhibited and subsequently results in chronic injury . many factors provoke an inflammatory response in the liver and are well known risk factors for liver cancer . liver cancer occurs during the progression of many chronic liver diseases associated with inflammation , such as hepatitis and steatohepatitis . hepatitis b virus ( hbv ) and hepatitis c virus ( hcv ) are found in approximately 80%90% of hepatocellular carcinoma ( hcc ) patients . chronic hbv infection is the dominant risk factor for liver cancers in asia and sub - saharan africa , particularly in china . hcv is a single - stranded rna virus that interacts with numerous proteins in hepatocytes and promotes the progression of hepatocarcinogenesis . chronic hcv infection is the dominant risk factor for liver cancers in japan , united states , and europe . cirrhosis contributes to hepatocarcinogenesis and is observed in most hcc patients with hbv or hcv infection . in addition to these viral infections , acetaldehyde and other metabolites generated by alcohol metabolism increase the amount of ros and reduce dna repair , which leads to hcc . epidemiological analysis revealed that alcohol consumption is positively associated with hcc incidence ; diabetes is another risk factor for hcc because epidemiologic studies reported that patients with type 2 diabetes have nearly 2.5-fold increased risk for hcc . recently , obesity was defined as a risk factor with an important role in hcc development . inflammation induced by viral or non - viral infection promotes the initiation and progression of hcc ; hcc develops in the background of chronic hepatitis . constitutive activation of these inflammatory signaling pathways , such as nf-b and jak / stat signaling pathways , occurs during the process of inflammation and is observed in various cancers . the first link between nf-b and cancer was revealed in a study showing that cancer cells were more sensitive to apoptosis - inducing stimuli after the nf-b signaling pathway was blocked . over the past decades , the diverse roles of nf-b signaling have been discovered in various types of carcinomas , including head and neck squamous cell carcinoma , leukemia , and cancers of breast , liver , prostate , colon , and lung . constitutive nf-b activation was observed in various cancers , where nf-b induces the expression of many pro - survival and anti - apoptotic factors , such as c - iap , xiap , and members of the bcl-2 family of proteins . analogously , the activation of nf-b might downregulate the expression of tumor suppressors , such as phosphatase and tensin homolog ( pten ) ; the downregulated expression increases akt signaling and suppresses the apoptosis of cancer cells . in addition to its pro - survival function , nf-b promotes cell cycle progression by inducing cyclin d1 and c - myc expression . nf-b signaling can also induce molecules such as vascular endothelial growth factor ( vegf ) and matrix metalloproteinases , and nf-b can promote angiogenesis and invasion by driving the expression of these proteins in many cancers . in 2004 , a group from israel confirmed that nf-b is an effective therapeutic target to treat hcc . hcc development in a mouse model was prevented through nf-b inhibition by a modified form of ib called super - repressor ib . another well - known signaling pathway that links inflammation and cancer is the janus kinase ( jak)/signal transducer and activator of transcription ( stat ) . stat3 , one of the most famous stat family members , was identified as a transcription factor that binds to the il-6-responsive element in hepatocytes . numerous studies reported that stat3 can also be activated by oncogenic proteins including ras or src . carcinogens , such as polychlorinated biphenyls , promotes liver cancer and regulates the activity of stat3 in hcc and normal liver cells . likewise , the constitutive activation of stat3 leads to the expression of bcl-2 family proteins essential for survival , such as survivin and ciap2 ; moreover , the apoptosis of cancer cells occurs when stat3 is downregulated in tumors . stat3 promotes the proliferation of cancer cells by inducing cyclin d1 and c - myc expression . in addition to molecules associated with proliferation and apoptotic inhibition , stat3 regulates the expression of the metalloproteinase family and vegf , consequently promoting angiogenesis and metastasis . stat3 is a key regulator of inflammation and cancer and thus might be an effective therapeutic target in inflammation - induced cancer . in 2006 , li et al . inhibited stat3 expression with antisense oligonucleotides in hcc cells and found that stat3 greatly reduced cell proliferation and migratory potential as well as induced apoptosis . mirnas constitute a class of short endogenous non - coding rnas that are highly conserved throughout evolution . the majority of mirnas are transcribed in the nucleus by rna polymerase ii as a primary mirna ( pri - mirna ) ; the typical pri - mirna contains stem - loop structures . furthermore , these stem - loop structures are cleaved into precursor mirnas ( pre - mirnas ) by drosha , an rna - specific ribonuclease enzyme complex . dicer functions in the biogenesis of mirnas as pre - mirnas are transported from the nucleus into the cytoplasm by exportin 5 . dicer is an endonuclease that specifically recognizes double - stranded rna complexes and transforms the pre - mirna into a 2022 nt mirna duplex . the mature mirna is incorporated into an rna - induced silencing complex ( risc ) through its interaction with ago proteins ; this mirna recognizes and binds to the 3-utr of target mrnas . the first mirna was discovered in caenorhabditis elegans in 1993 and was identified as an essential regulator of postembryonic developmental events . from that first discovery , currently , mirnas are recognized as key mediators influencing nearly all biological processes , including development , immune response , differentiation , proliferation , apoptosis , invasion , and metastasis . in addition , the expression of numerous mirnas is dysregulated during nearly all pathological processes . mirnas gained a widely accepted role in initiation and progression of various human cancers , including hcc ; evidence indicated that these mirnas act as oncogenes or tumor suppressors . . some of these mirnas might be significant to diagnosis or prognosis of hcc patients . accumulating evidence revealed that mirnas are a novel class of molecules linking inflammation and hcc . chronic hepatitis virus infection leads to liver inflammation , damage , fibrosis , cell regeneration , and malignant transformation ( hcc ) . many mirnas play a crucial role in the regulation of hepatitis virus - induced chronic infla - mmation and hcc ( table 1 ) . for instance , mir-27 is upregulated by hcv infection and can promote cell migration and invasion by suppressing spry2 in hcc cells . mir-146a expression is consistently increased in hcv - infected cells , primary hepatocytes , and liver tissue from hcv - infected patients . increased mir-146a level promotes viral infection and is associated with the pathogenesis of liver disease . in the following section , we will summarize the key roles of relevant mirnas in hepatitis virus infection and hcc . mirnas associated with hepatitis virus infection and hcc the first mirna discovered in humans is let-7 , which is highly conserved across animal species and promotes differentiation . recent studies revealed that let-7 is frequently downregulated in many cancers , such as breast , ovarian , prostate , lung , and liver cancers . the hbv x protein ( hbx ) encoded by the hbv genome is a multifunctional transactivator that plays crucial roles in hbv - associated hepatocarcinogenesis . hbx lacks a dna binding domain and acts as a trans - coactivator through interaction with other cellular transcription factors , by which hbx might regulate let-7 family members . in addition to hbx induction , hbv pres2 rna can sequester let-7 g , which in turn reduces intrinsic let-7 g functions in hcc . wang et al . concluded that hbx - mediated down - regulation of let-7 might be responsible for reduced let-7 expression in hbv - associated hcc . mirnas from the let-7 family act as tumor suppressors in hcc by inhibiting proliferation and invasion , as well as inducing apoptosis by targeting many oncogenes including high mobility group proteins , bcl - xl , stat3 , and c - myc . mir-122 is the most abundant mirna expressed in the liver , which accounts for 70% of the total mirnas in an adult liver . the expression of mir-122 is increased in the liver during embryonic development , and the first identified target of this mirna is cationic amino acid transporter 1 . in recent years , increasing evidence suggested that mir-122 plays an important role in initiation and progression of hcc . li et al . found that mir-122 expression is low in patients with chronic hepatitis b and hcc tumors and has negative correlation with fibrotic stage , age , and liver transaminase levels . the downregulation of mir-122 in patients with chronic hepatitis b and hcc tissues is affected by hbv infection . ppar was identified as a transcription factor regulating mir-122 expression ; the underlying cause of hbx - induced mir-122 downregulation might be the direct interaction between hbx and ppar , which blocks ppar nuclear translocation . in 2013 , li et al . further reported that all four hbv mrnas contain a binding site for mir-122 and act as a mirna sponge sequestering mir-122 in hbv - infected cells . in addition , mir-122 knockout mice will possibly develop hepatitis , fibrosis , and hcc . the reduced mir-122 expression in hcc tissues is characterized by a poor prognosis and consistent with a suppressed hepatic phenotype and an increased metastatic potential . moreover , mir-122 expression can be suppressed by two inflammatory cytokines , il-6 and tnf- , which downregulate the mir-122 transcription factors c / ebp and hnf3 and induce c - myc - mediated c / ebp inhibition in chronic hepatic inflammation . mir-122 downregulation results in the upregulation of target genes that promote hepatocarcinogenesis , including pituitary tumor trans - forming gene binding factor ( pbf ) , cyclin g1 , pkm2 , adam10 , adam17 , srf , and pkr activator . mir-101 downregulation was reported in various cancers , including hcc , suggesting that mir-101 functions as a tumor suppressor . reduced mir-101 expression in hcc tissues is associated with advanced tumor progression and poor survival in hcc patients ; the serum mir-101 level might be a potential hcc biomarker . overexpression of mir-101 sensitizes the hcc cells to apoptosis induced by serum deprivation and chemotherapeutic drugs . mcl-1 , a member of the bcl-2 family , is a target of mir-101 and sensitizes cancer cells to apoptosis . mir-101 enhances cisplatin - induced apoptosis by targeting stmn1 , rab5a , and mtor , as well as suppresses epithelial - mesenchymal transition ( emt ) in hepatocytes via the regulation of zeb1 expression , which results in e - cadherin down - regulation . in 2010 , the first link between mir-155 and hepatitis viruses was based on the observation that mir-155 precursor ( termed bic ) is responsible for hcv infection . after patients with hepatitis c were treated with interferon alpha and ribavirin , the lowest bic expression was detected in patients who lacked hcv rna in both serum and peripheral blood mononuclear cells . previously , mir-155 was identified as an onco - mir in various cancers , including lung , breast , stomach , and prostate cancers . a mouse model revealed that mir-155 induced by inflammation could promote autoimmune inflammation by enhancing inflammatory t cell development . mir-155 a positive correlation was observed between mir-155 expression and ip-10 levels in the plasma , which is widely considered a marker of liver inflammation degree . upon nf-b activation by hcv - rna infection the upregulation of mir-155 in cases of hepatitis and hcc promotes hepatocyte proliferation and hcc tumor growth by activating the wnt/-catenin signaling pathway . in addition to hcc pathogenesis during chronic viral infections such as hcv , the upregulation of mir-155 was also observed in the pathogenesis of non - alcoholic steatohepatitis ( nash)-associated hcc . however , similar to hcv - induced mir-155 , the activation of nf-b signaling pathway plays a key role in mir-155 expression in hcc and other hepatocyte - derived cells . in a mouse model of nash , mir-155 downregulated the tumor suppressor target c / ebp , thus promoting liver pathogenesis . mir-21 is a well - established onco - mir that is upregulated in various cancers , including lung , breast , prostate , colon , stomach , pancreatic , and liver cancers . the relationship between mir-21 and inflammation was observed in uv - exposed skin , where mir-21 was activated by ppar via the tgf- signaling pathway . the upregulation of mir-21 was reported in other inflammation - associated tissues and cancers , such as allergy - induced airway inflammation , gastric cancer , pre - b - cell lymphoma , and colorectal cancer . the relevance of mir-21 and inflammation in liver tissues was established by associating biomarkers with hcc diagnosis and prognosis . mir-21 and mir-122 were analyzed in tissues obtained by needle biopsies from both hcv - infected and non - infected patients . the expression of mir-21 was positively correlated with fibrotic stage in patients with chronic hepatitis c ; mir-21 promotes liver fibrosis by targeting the fibrosis inhibitor smad7 . in serum in addition , one study reported that hbx increases the expression of mir-21 and subsequently promotes the progression of hcc by targeting pten . in another study , hbx - mediated mir-21 upregulation inhibits expression of the tumor suppressor pdcd4 in hcc cells . mir-221 is another onco - mir that is upregulated in cancers such as breast , prostate , glioma , and liver cancers . moreover , the expression of mir-221 in later stages of hcc is higher than that in early stages . therefore , mir-221 is a poor prognosis risk factor because it was increased in mouse models of liver fibrosis and upregulated in the human liver in a fibrosis progression - dependent manner . hbx promotes the proliferation of hcc cells and upregulates the expression of mir-221 during both hbv infection and hcc . several tumor - related genes were reported to be the targets of mir-221 ; among these genes , p27kip1 is well - recognized . the expression of mir-221/p27kip1 axis was confirmed in glioblastomas , breast , lung , and liver cancers . other than p27kip1 , many mir-221 targets were reported , such as hdac6 , pten , and tissue inhibitor of metalloproteinase-3 ( timp3 ) . in mouse models of hcc , tumor - bearing mice that were treated with chol - anti - mir-221 mir-221 is a potential target for hcc treatment and functions by inhibiting the progression of hcc and promoting survival . chronic inflammation can facilitate the development and progression of hcc by the constitutive activation of inflammation signaling pathways . inhibition of these signaling pathways would be a novel therapeutic strategy to suppress this inflammation - associated process . many mirnas were reported to regulate the inflammatory signaling mediated by nf-b and jak / stat3 , among others , in hcc ( table 2 ) . mir-195 , mir-194 , and mir-127 suppressed nf-b activation by directly or indirectly targeting nf-b signaling ; these mirnas also function as tumor suppressors in hcc . mir-219 is reduced in hcc where it functions as a tumor suppressor ; this mirna might suppress jak / stat3 signaling via direct targeting of structural maintenance of chromosome 4 protein ( smc4 ) . mir-637 is a tumor suppressor that inhibits cell growth and induces apoptosis by suppressing stat3 activation in hcc . mirnas as regulators of inflammatory signaling in hcc mir-26a was suggested to play an important role in cirrhosis and inflammation - induced hcc . reduced mir-26a expression was detected in hcc tissues ; the decreased expression in hcc tissues might be an independent predictor of poor survival . clinical analysis revealed that the expression of mir-26a is negatively correlated with il-6 mrna and il-6 protein level . as il-6 is a direct target of mir-26a , the mrna and protein levels of this cytokine are significantly reduced by mir-26a overexpression in hcc cells . mir-26a functions as a tumor suppressor that inhibits tumor growth and metastasis , partly through the il - stat3 signaling pathway . m - csf expression was also reported as a predictor of frequent metastasis and poor survival ; mir-26a affects macrophage polarization and inhibits macrophage recruitment by down - regulating m - csf expression . in addition , hcc cells transfected with mir-126b exhibit significantly reduced nf-b reporter activity in response to tnf. two well - known upstream regulators of nf-b signaling pathway , tak1 and tab 3 , were identified as direct targets of mir-26b . mir-26b prevents doxorubicin - induced nf-b activation and promotes apoptosis in hcc cells . in one study , mir-451 was often downregulated in primary hcc tissues ; reduced mir-451 expression was significantly associated with advanced tnm stage , lymph node metastasis , vascular invasion , and poor survival , indicating that mir-451 might act as a tumor suppressor in hcc . mir-451 might inhibit hcc tumorigenesis and metastasis by down - regulating cyclin d1 and c - myc at both mrna and protein levels . further study indicated that mir-451 overexpression affected the nf-b nuclear translocation and significantly decreased nf-b activity by directly targeting ikk. as expected , the inhibition of mir-451 significantly increased nf-b activity in hcc cells . moreover , a recent study revealed that reduced expression of the il-6 receptor was observed in cells that overexpressed mir-451 , and that il-6 receptor was a direct target of mir-451 . mir-451 reduced vegf production in hcc cells by targeting the il-6/stat3 signaling pathway . mir-124 is a well - known tumor suppressor that is frequently downregulated in various cancers . numerous studies revealed that mir-124 inhibits inflammatory signaling by targeting stat3 in different cancer types including glioblastoma , gastric cancer , endometrial cancer , colon cancer , and hcc . recently , nf-b p65 was reported to be a direct mir-124 target in b - cell lymphomas . the downregulation of mir-124 in b - cell lymphomas enhances the nf-b signaling pathway and upregulates genes , such as myc and bcl-2 , associated with proliferation , anti - apoptosis , metastasis , and pro - survival . stat3 was identified as a direct mir-124 target in hcc ; one study found that mir-124 inhibited cell proliferation and induced apoptosis in hcc cells by targeting stat3 . moreover , an hnf4-nf-b feedback circuit that includes mir-124 was identified in hcc . reduced hnf4 could be a biomarker for poor prognosis , as hnf4 suppresses hcc metastasis by inhibiting nf-b activation . mir-124 also plays an important role in rela suppression by hnf4. mir-124 was identified as an hnf4 target that binds to the 3utr of rela . mir-124 we discovered that mir-125b was significantly downregulated in hcc , could be used as a biomarker for prolonged hcc patient survival , and functions as a tumor suppressor . mir-125b inhibits the proliferation of hcc cells by inducing cell cycle arrest during g1/s transition via its target gene , lin28b . il-6r was characterized as the subunit that binds to il-6 ; the binding of il-6 to il-6r activates the jak / stat3 signaling pathway . mir-125b can block the il-6-induced phosphory - lation and nuclear translocation of stat3 in hcc cells , which in turn promotes hcc apoptosis . in addition , mir-125b serves as a tumor suppressor that inhibits migration and invasion of hcc cells by targeting the transcriptional co - activator with pdz - binding motif ( taz ) . several studies revealed that mir-370 acts as tumor promoter in gastric cancer , prostate cancer , and aml ; whereas other studies reported mir-370 as a tumor suppressor in leukemia and oral squamous carcinoma . xu et al . observed that the down - regulation of mir-370 was associated with hcc development in rats and correlated with poor survival in hcc patients , indicating that mir-370 serves as a tumor suppressor in hcc . the restoration of mir-370 inhibits proliferation , migration , and invasion of hcc cells and suppresses tumorigenesis of hcc by directly targeting lin28a . lin28a is a post - transcriptional modulator of mrnas that can directly bind to p65 mrna . mir-370 represses nf-b activation and inhibits the migration and invasion of hcc cells through downregulation of lin28a . sun et al . confirmed the tumor suppressor role of mir-370 in hcc cells , showing that mir-370 inhibited hcc cell proliferation and colony formation ability by activating the pi3k / akt signaling pathway . in addition to mirnas that inhibit inflammatory signaling , some mirnas , such as mir-155 , mir-362 , mir-301a , and mir-1180 , promote the inflammatory response by repressing negative regulators of inflammatory signaling . the nf-b signaling pathway is activated by mir-1180 overexpression , which occurs through the downregulation of mir-1180 direct targets , otud7b and tnip2 . another study confirmed the expression of tnip2 , a direct target of mir-1180 , in hcc cells . the ectopic expression of mir-1180 promoted the proliferation of hcc cells and inhibited apoptosis because nf-b induces the expression of genes that participate in pro - survival and anti - apoptotic processes . ni et al . demonstrated that mir-362 - 5p , which is frequently upregulated in hcc , promoted nf-b signaling by downregulating cyld ; cyld is a protein that binds to nf-b essential modulator and functions as a negative regulator of nf-b signaling . these inflammatory pathways and related transcription factors can affect the development of hcc by regulating mirna expression . several studies confirmed that inflammatory signaling facilitates the development of hcc through the downregulation or upregulation of mirnas in cells ( table 3 ) . mirnas as mediators of inflammatory signaling in hcc ning et al . observed feedback regulation of hnf4 and nf-b in hcc . their study revealed that hnf4 dramatically suppresses the nf-b signaling pathway and that rela overexpression or knockdown decreases or increases the expression of hnf4 , respectively . during chronic hepatic inflammation , nf-b induced the expression of mir-21 , which represses hnf4 expression and contributes to hepatocyte de - differentiation and hepatocarcinogenesis . in addition to direct induction of mir-21 expression by nf-b , mir-21 transcriptional activity is enhanced by high - mobility group box-1 protein ( hmgb1 ) , an important participant in inflammation . hmgb1 is a conserved nuclear protein that functions as a chromatin binding factor and allows transcription factors to access specific dna binding sites . hmgb1 enhances p65/p50 or p50/p50 binding to specific dna sites rather than the binding of p65/p65 , c - rel / c - rel , p65/c - rel , and p50/c - rel . given its role in the inflammatory cascade , hmgb1 is upregulated in hcc and induces mir-21 expression in hcc cells . the hmgb1/mir-21 signaling axis inhibits the downstream targets reck and timp3 as well as promotes cell proliferation , invasion , and migration . mir-143 is upregulated in hcc tissues , indicating that mir-143 serves as a tumor promoter . mir-143 expression was increased by hbx - mediated nf-b activation , although metastasis will be suppressed by mir-143 inhibition . nf-b activates transcription by specifically binding to the putative mir-143 promoter , as the nf-b binding site identifies 5.06 kb upstream of mir-143 . mir-143 promotes cell invasion and tumor metastasis by suppressing the direct target gene fndc3b . moreover , mir-143 inhibition results in apoptosis of hcc cells and suppression of hcc tumorigenesis . in addition , mir-143 was upregulated in serum samples from patients with chronic hepatitis and hcc and therefore might be a useful biomarker for both diseases . in addition to the upregulation of mir-221 by hbv , mir-221 can also be induced by hcv infection in an nf-b - dependent manner . however , the increased expression of mir-221 was blocked after treatment with pdtc , an nf-b inhibitor . this finding suggests that the hcv - induced upregulation of mir-221 requires nf-b activation and that nf-b signaling pathway plays an important role in inflammation - associated cancer . the expression of mir-224 was upregulated in both cirrhotic livers and hcc samples , suggesting the use of mir-224 as a biomarker of liver inflammation and hcc . the upregulation of mir-224 begins in the pre - cancerous stage and continues during hcc development . in one study in addition , mir-224 levels were significantly higher in patients with advanced hcc stages than in patients with early stages . mir-224 could be induced by a set of pro - inflammatory factors , such as tnf , lps , and lt , suggesting that nf-b signaling regulates mir-224 . inflammation - mediated activation of mir-224 promotes hcc cell migration and invasion by targeting rho gtpase - activating proteins . as an onco - mir , the overexpression of mir-224 also facilitates the migration and invasion of hcc cells by targeting hoxd10 and promotes proliferation by targeting p21 . mir-197 is well - studied during the progression of various cancers in which it plays a role in apoptosis , proliferation , angiogenesis , metastasis , and drug resistance . a negative correlation between mir-197 expression and il-6 and stat3 protein levels was observed in hcc tissues , indicating that mir-197 is involved in the stat3 signaling pathway . il-6 is a well - known pro - inflammatory factor that is upregulated during an inflammatory state . the expression of mature mir-197 was reduced by il-6 stimulation , whereas the expression of pri - mir-197 remained unchanged by il-6 stimulation in hcc cells . further investigation revealed that drosha binding to pri - mir-197 decreased after il-6 stimulation , indicating that il-6 downregulated mir-197 in a post - transcriptional manner by inhibiting drosha binding to pri - mir-197 . moreover , stat3 was identified as a direct target of mir-197 . as a tumor suppressor , mir-197 inhibits proliferation and invasiveness of hcc cells as well as suppresses the tumorigenesis of hcc xenografts . the first evidence of the role of mir-23a in hcc development was revealed in 2008 . mir-23a induced by tgf is remarkably increased in hcc tissues and functions as an anti - apoptotic and pro - proliferative factor in hcc cells . later on , wang et al . found that mir-23 can be induced by il-6 and stat3 signaling , as a potential stat3 binding site was identified in the mir-23a promoter region . stat3-activated mir-23a downregulates the target genes g6pc and pcg - a and suppresses gluconeogenesis ; this reduction in gluconeogenesis facilitates hcc tumorigenesis . chronic hepatitis virus infection leads to liver inflammation , damage , fibrosis , cell regeneration , and malignant transformation ( hcc ) . many mirnas play a crucial role in the regulation of hepatitis virus - induced chronic infla - mmation and hcc ( table 1 ) . for instance , mir-27 is upregulated by hcv infection and can promote cell migration and invasion by suppressing spry2 in hcc cells . mir-146a expression is consistently increased in hcv - infected cells , primary hepatocytes , and liver tissue from hcv - infected patients . increased mir-146a level promotes viral infection and , we will summarize the key roles of relevant mirnas in hepatitis virus infection and hcc . mirnas associated with hepatitis virus infection and hcc the first mirna discovered in humans is let-7 , which is highly conserved across animal species and promotes differentiation . recent studies revealed that let-7 is frequently downregulated in many cancers , such as breast , ovarian , prostate , lung , and liver cancers . the hbv x protein ( hbx ) encoded by the hbv genome is a multifunctional transactivator that plays crucial roles in hbv - associated hepatocarcinogenesis . hbx lacks a dna binding domain and acts as a trans - coactivator through interaction with other cellular transcription factors , by which hbx might regulate let-7 family members . in addition to hbx induction , hbv pres2 rna can sequester let-7 g , which in turn reduces intrinsic let-7 g functions in hcc . wang et al . concluded that hbx - mediated down - regulation of let-7 might be responsible for reduced let-7 expression in hbv - associated hcc . mirnas from the let-7 family act as tumor suppressors in hcc by inhibiting proliferation and invasion , as well as inducing apoptosis by targeting many oncogenes including high mobility group proteins , bcl - xl , stat3 , and c - myc . mir-122 is the most abundant mirna expressed in the liver , which accounts for 70% of the total mirnas in an adult liver . the expression of mir-122 is increased in the liver during embryonic development , and the first identified target of this mirna is cationic amino acid transporter 1 . in recent years , increasing evidence suggested that mir-122 plays an important role in initiation and progression of hcc . li et al . found that mir-122 expression is low in patients with chronic hepatitis b and hcc tumors and has negative correlation with fibrotic stage , age , and liver transaminase levels . the downregulation of mir-122 in patients with chronic hepatitis b and hcc tissues is affected by hbv infection . ppar was identified as a transcription factor regulating mir-122 expression ; the underlying cause of hbx - induced mir-122 downregulation might be the direct interaction between hbx and ppar , which blocks ppar nuclear translocation . in 2013 , li et al . further reported that all four hbv mrnas contain a binding site for mir-122 and act as a mirna sponge sequestering mir-122 in hbv - infected cells . in addition , mir-122 knockout mice will possibly develop hepatitis , fibrosis , and hcc . the reduced mir-122 expression in hcc tissues is characterized by a poor prognosis and consistent with a suppressed hepatic phenotype and an increased metastatic potential . moreover , mir-122 expression can be suppressed by two inflammatory cytokines , il-6 and tnf- , which downregulate the mir-122 transcription factors c / ebp and hnf3 and induce c - myc - mediated c / ebp inhibition in chronic hepatic inflammation . mir-122 downregulation results in the upregulation of target genes that promote hepatocarcinogenesis , including pituitary tumor trans - forming gene binding factor ( pbf ) , cyclin g1 , pkm2 , adam10 , adam17 , srf , and pkr activator . mir-101 downregulation was reported in various cancers , including hcc , suggesting that mir-101 functions as a tumor suppressor . reduced mir-101 expression in hcc tissues is associated with advanced tumor progression and poor survival in hcc patients ; the serum mir-101 level might be a potential hcc biomarker . overexpression of mir-101 sensitizes the hcc cells to apoptosis induced by serum deprivation and chemotherapeutic drugs . mcl-1 , a member of the bcl-2 family , is a target of mir-101 and sensitizes cancer cells to apoptosis . mir-101 enhances cisplatin - induced apoptosis by targeting stmn1 , rab5a , and mtor , as well as suppresses epithelial - mesenchymal transition ( emt ) in hepatocytes via the regulation of zeb1 expression , which results in e - cadherin down - regulation . in 2010 , the first link between mir-155 and hepatitis viruses was based on the observation that mir-155 precursor ( termed bic ) is responsible for hcv infection . after patients with hepatitis c were treated with interferon alpha and ribavirin , the lowest bic expression was detected in patients who lacked hcv rna in both serum and peripheral blood mononuclear cells . previously , mir-155 was identified as an onco - mir in various cancers , including lung , breast , stomach , and prostate cancers . a mouse model revealed that mir-155 induced by inflammation could promote autoimmune inflammation by enhancing inflammatory t cell development . mir-155 a positive correlation was observed between mir-155 expression and ip-10 levels in the plasma , which is widely considered a marker of liver inflammation degree . upon nf-b activation by hcv - rna infection the upregulation of mir-155 in cases of hepatitis and hcc promotes hepatocyte proliferation and hcc tumor growth by activating the wnt/-catenin signaling pathway . in addition to hcc pathogenesis during chronic viral infections such as hcv , the upregulation of mir-155 was also observed in the pathogenesis of non - alcoholic steatohepatitis ( nash)-associated hcc . however , similar to hcv - induced mir-155 , the activation of nf-b signaling pathway plays a key role in mir-155 expression in hcc and other hepatocyte - derived cells . in a mouse model of nash , mir-155 downregulated the tumor suppressor target c / ebp , thus promoting liver pathogenesis . mir-21 is a well - established onco - mir that is upregulated in various cancers , including lung , breast , prostate , colon , stomach , pancreatic , and liver cancers . the relationship between mir-21 and inflammation was observed in uv - exposed skin , where mir-21 was activated by ppar via the tgf- signaling pathway . the upregulation of mir-21 was reported in other inflammation - associated tissues and cancers , such as allergy - induced airway inflammation , gastric cancer , pre - b - cell lymphoma , and colorectal cancer . the relevance of mir-21 and inflammation in liver tissues was established by associating biomarkers with hcc diagnosis and prognosis . mir-21 and mir-122 were analyzed in tissues obtained by needle biopsies from both hcv - infected and non - infected patients . the expression of mir-21 was positively correlated with fibrotic stage in patients with chronic hepatitis c ; mir-21 promotes liver fibrosis by targeting the fibrosis inhibitor smad7 . in serum in addition , one study reported that hbx increases the expression of mir-21 and subsequently promotes the progression of hcc by targeting pten . in another study , hbx - mediated mir-21 upregulation inhibits expression of the tumor suppressor pdcd4 in hcc cells . mir-221 is another onco - mir that is upregulated in cancers such as breast , prostate , glioma , and liver cancers . moreover , the expression of mir-221 in later stages of hcc is higher than that in early stages . therefore , mir-221 is a poor prognosis risk factor because it was increased in mouse models of liver fibrosis and upregulated in the human liver in a fibrosis progression - dependent manner . hbx promotes the proliferation of hcc cells and upregulates the expression of mir-221 during both hbv infection and hcc . several tumor - related genes were reported to be the targets of mir-221 ; among these genes , p27kip1 is well - recognized . the expression of mir-221/p27kip1 axis was confirmed in glioblastomas , breast , lung , and liver cancers . other than p27kip1 , many mir-221 targets were reported , such as hdac6 , pten , and tissue inhibitor of metalloproteinase-3 ( timp3 ) . in mouse models of hcc , tumor - bearing mice that were treated with chol - anti - mir-221 have a survival advantage compared with mice in the control group . mir-221 is a potential target for hcc treatment and functions by inhibiting the progression of hcc and promoting survival . chronic inflammation can facilitate the development and progression of hcc by the constitutive activation of inflammation signaling pathways . inhibition of these signaling pathways would be a novel therapeutic strategy to suppress this inflammation - associated process . many mirnas were reported to regulate the inflammatory signaling mediated by nf-b and jak / stat3 , among others , in hcc ( table 2 ) . mir-195 , mir-194 , and mir-127 suppressed nf-b activation by directly or indirectly targeting nf-b signaling ; these mirnas also function as tumor suppressors in hcc . mir-219 is reduced in hcc where it functions as a tumor suppressor ; this mirna might suppress jak / stat3 signaling via direct targeting of structural maintenance of chromosome 4 protein ( smc4 ) . mir-637 is a tumor suppressor that inhibits cell growth and induces apoptosis by suppressing stat3 activation in hcc . mirnas as regulators of inflammatory signaling in hcc mir-26a was suggested to play an important role in cirrhosis and inflammation - induced hcc . reduced mir-26a expression was detected in hcc tissues ; the decreased expression in hcc tissues might be an independent predictor of poor survival . clinical analysis revealed that the expression of mir-26a is negatively correlated with il-6 mrna and il-6 protein level . as il-6 is a direct target of mir-26a , the mrna and protein levels of this cytokine are significantly reduced by mir-26a overexpression in hcc cells . mir-26a functions as a tumor suppressor that inhibits tumor growth and metastasis , partly through the il - stat3 signaling pathway . m - csf expression was also reported as a predictor of frequent metastasis and poor survival ; mir-26a affects macrophage polarization and inhibits macrophage recruitment by down - regulating m - csf expression . in addition , hcc cells transfected with mir-126b exhibit significantly reduced nf-b reporter activity in response to tnf. two well - known upstream regulators of nf-b signaling pathway , tak1 and tab 3 , were identified as direct targets of mir-26b . mir-26b prevents doxorubicin - induced nf-b activation and promotes apoptosis in hcc cells . in one study , mir-451 was often downregulated in primary hcc tissues ; reduced mir-451 expression was significantly associated with advanced tnm stage , lymph node metastasis , vascular invasion , and poor survival , indicating that mir-451 might act as a tumor suppressor in hcc . mir-451 might inhibit hcc tumorigenesis and metastasis by down - regulating cyclin d1 and c - myc at both mrna and protein levels . further study indicated that mir-451 overexpression affected the nf-b nuclear translocation and significantly decreased nf-b activity by directly targeting ikk. as expected , the inhibition of mir-451 significantly increased nf-b activity in hcc cells . moreover , a recent study revealed that reduced expression of the il-6 receptor was observed in cells that overexpressed mir-451 , and that il-6 receptor was a direct target of mir-451 . mir-451 reduced vegf production in hcc cells by targeting the il-6/stat3 signaling pathway . mir-124 is a well - known tumor suppressor that is frequently downregulated in various cancers . numerous studies revealed that mir-124 inhibits inflammatory signaling by targeting stat3 in different cancer types including glioblastoma , gastric cancer , endometrial cancer , colon cancer , and hcc . recently , nf-b p65 was reported to be a direct mir-124 target in b - cell lymphomas . the downregulation of mir-124 in b - cell lymphomas enhances the nf-b signaling pathway and upregulates genes , such as myc and bcl-2 , associated with proliferation , anti - apoptosis , metastasis , and pro - survival . stat3 was identified as a direct mir-124 target in hcc ; one study found that mir-124 inhibited cell proliferation and induced apoptosis in hcc cells by targeting stat3 . moreover , an hnf4-nf-b feedback circuit that includes mir-124 was identified in hcc . reduced hnf4 could be a biomarker for poor prognosis , as hnf4 suppresses hcc metastasis by inhibiting nf-b activation . mir-124 also plays an important role in rela suppression by hnf4. mir-124 was identified as an hnf4 target that binds to the 3utr of rela . mir-124 we discovered that mir-125b was significantly downregulated in hcc , could be used as a biomarker for prolonged hcc patient survival , and functions as a tumor suppressor . mir-125b inhibits the proliferation of hcc cells by inducing cell cycle arrest during g1/s transition via its target gene , lin28b . il-6r was characterized as the subunit that binds to il-6 ; the binding of il-6 to il-6r activates the jak / stat3 signaling pathway . mir-125b can block the il-6-induced phosphory - lation and nuclear translocation of stat3 in hcc cells , which in turn promotes hcc apoptosis . in addition , mir-125b serves as a tumor suppressor that inhibits migration and invasion of hcc cells by targeting the transcriptional co - activator with pdz - binding motif ( taz ) . several studies revealed that mir-370 acts as tumor promoter in gastric cancer , prostate cancer , and aml ; whereas other studies reported mir-370 as a tumor suppressor in leukemia and oral squamous carcinoma . xu et al . observed that the down - regulation of mir-370 was associated with hcc development in rats and correlated with poor survival in hcc patients , indicating that mir-370 serves as a tumor suppressor in hcc . the restoration of mir-370 inhibits proliferation , migration , and invasion of hcc cells and suppresses tumorigenesis of hcc by directly targeting lin28a . lin28a is a post - transcriptional modulator of mrnas that can directly bind to p65 mrna . mir-370 represses nf-b activation and inhibits the migration and invasion of hcc cells through downregulation of lin28a . sun et al . confirmed the tumor suppressor role of mir-370 in hcc cells , showing that mir-370 inhibited hcc cell proliferation and colony formation ability by activating the pi3k / akt signaling pathway . in addition to mirnas that inhibit inflammatory signaling , some mirnas , such as mir-155 , mir-362 , mir-301a , and mir-1180 , promote the inflammatory response by repressing negative regulators of inflammatory signaling . the nf-b signaling pathway is activated by mir-1180 overexpression , which occurs through the downregulation of mir-1180 direct targets , otud7b and tnip2 . another study confirmed the expression of tnip2 , a direct target of mir-1180 , in hcc cells . the ectopic expression of mir-1180 promoted the proliferation of hcc cells and inhibited apoptosis because nf-b induces the expression of genes that participate in pro - survival and anti - apoptotic processes . ni et al . demonstrated that mir-362 - 5p , which is frequently upregulated in hcc , promoted nf-b signaling by downregulating cyld ; cyld is a protein that binds to nf-b essential modulator and functions as a negative regulator of nf-b signaling . these inflammatory pathways and related transcription factors can affect the development of hcc by regulating mirna expression . several studies confirmed that inflammatory signaling facilitates the development of hcc through the downregulation or upregulation of mirnas in cells ( table 3 ) . mirnas as mediators of inflammatory signaling in hcc ning et al . observed feedback regulation of hnf4 and nf-b in hcc . their study revealed that hnf4 dramatically suppresses the nf-b signaling pathway and that rela overexpression or knockdown decreases or increases the expression of hnf4 , respectively . during chronic hepatic inflammation , nf-b induced the expression of mir-21 , which represses hnf4 expression and contributes to hepatocyte de - differentiation and hepatocarcinogenesis . in addition to direct induction of mir-21 expression by nf-b , mir-21 transcriptional activity is enhanced by high - mobility group box-1 protein ( hmgb1 ) , an important participant in inflammation . hmgb1 is a conserved nuclear protein that functions as a chromatin binding factor and allows transcription factors to access specific dna binding sites . hmgb1 enhances p65/p50 or p50/p50 binding to specific dna sites rather than the binding of p65/p65 , c - rel / c - rel , p65/c - rel , and p50/c - rel . given its role in the inflammatory cascade , hmgb1 is upregulated in hcc and induces mir-21 expression in hcc cells . the hmgb1/mir-21 signaling axis inhibits the downstream targets reck and timp3 as well as promotes cell proliferation , invasion , and migration . mir-143 is upregulated in hcc tissues , indicating that mir-143 serves as a tumor promoter . mir-143 expression was increased by hbx - mediated nf-b activation , although metastasis will be suppressed by mir-143 inhibition . nf-b activates transcription by specifically binding to the putative mir-143 promoter , as the nf-b binding site identifies 5.06 kb upstream of mir-143 . mir-143 promotes cell invasion and tumor metastasis by suppressing the direct target gene fndc3b . moreover , mir-143 inhibition results in apoptosis of hcc cells and suppression of hcc tumorigenesis . in addition , mir-143 was upregulated in serum samples from patients with chronic hepatitis and hcc and therefore might be a useful biomarker for both diseases . in addition to the upregulation of mir-221 by hbv , mir-221 can also be induced by hcv infection in an nf-b - dependent manner . however , the increased expression of mir-221 was blocked after treatment with pdtc , an nf-b inhibitor . this finding suggests that the hcv - induced upregulation of mir-221 requires nf-b activation and that nf-b signaling pathway plays an important role in inflammation - associated cancer . the expression of mir-224 was upregulated in both cirrhotic livers and hcc samples , suggesting the use of mir-224 as a biomarker of liver inflammation and hcc . the upregulation of mir-224 begins in the pre - cancerous stage and continues during hcc development . in one study , patients with lower mir-224 expression exhibited a favorable trend of survival . in addition , mir-224 levels were significantly higher in patients with advanced hcc stages than in patients with early stages . mir-224 could be induced by a set of pro - inflammatory factors , such as tnf , lps , and lt , suggesting that nf-b signaling regulates mir-224 . inflammation - mediated activation of mir-224 promotes hcc cell migration and invasion by targeting rho gtpase - activating proteins . as an onco - mir , the overexpression of mir-224 also facilitates the migration and invasion of hcc cells by targeting hoxd10 and promotes proliferation by targeting p21 . mir-197 is well - studied during the progression of various cancers in which it plays a role in apoptosis , proliferation , angiogenesis , metastasis , and drug resistance . a negative correlation between mir-197 expression and il-6 and stat3 protein levels was observed in hcc tissues , indicating that mir-197 is involved in the stat3 signaling pathway . il-6 is a well - known pro - inflammatory factor that is upregulated during an inflammatory state . the expression of mature mir-197 was reduced by il-6 stimulation , whereas the expression of pri - mir-197 remained unchanged by il-6 stimulation in hcc cells . further investigation revealed that drosha binding to pri - mir-197 decreased after il-6 stimulation , indicating that il-6 downregulated mir-197 in a post - transcriptional manner by inhibiting drosha binding to pri - mir-197 . moreover , stat3 was identified as a direct target of mir-197 . as a tumor suppressor , mir-197 inhibits proliferation and invasiveness of hcc cells as well as suppresses the tumorigenesis of hcc xenografts . the first evidence of the role of mir-23a in hcc development was revealed in 2008 . mir-23a induced by tgf is remarkably increased in hcc tissues and functions as an anti - apoptotic and pro - proliferative factor in hcc cells . later on , wang et al . found that mir-23 can be induced by il-6 and stat3 signaling , as a potential stat3 binding site was identified in the mir-23a promoter region . stat3-activated mir-23a downregulates the target genes g6pc and pcg - a and suppresses gluconeogenesis ; this reduction in gluconeogenesis facilitates hcc tumorigenesis . mirnas have a well - recognized role in liver inflammation and hepatocarcinogenesis ; many mirnas are dysregulated during chronic liver inflammation and hcc progression . mirnas were regarded as novel targets for hcc therapy ; the first study using mirna - based therapy was conducted in 2005 . in that study , mir-122 expression was silenced by antagomirs , which act as efficient and specific silencers of endogenous mirnas . a clinical trial of treating hcv infection by mir-122 inhibition was performed because mir-122 targets hcv genomic rna and promotes hcv viral translation . jong - kook park and colleagues treated tumor - bearing mice with chol - anti - mir-221 or a cholesterol - labeled control oligo . consistent with the in vitro results , improved survival was observed in the group of mice treated with chol - anti - mir-221 . in addition , mir-21 is a tumor promoter and functions as a prognostic factor , as previously described . mir-21 promotes the proliferation , metastasis , and tumorigenesis of hcc and was validated as an onco - mir involved in drug resistance . mir-21 suppression by lenti - mir-21-i combined with 5-fluorouracil and pirarubicin treatment leads to significant suppression of hcc cell proliferation ; 5-fluorouracil and pirarubicin downregulated the mir-21 expression through ap-1 signaling . tumor - bearing nude mice were intra - tumorally injected with either mir-199 adenovirus or pbs as a control . these results were confirmed in a transgenic model in which mice were predisposed to hcc development . the group of mice treated with mir-199 adenovirus exhibited a reduced tumor burden and a decreased number of tumor nodules compared with the control group . it can be expected in the future that any mirnas acting as tumor suppressors can be applied as therapeutic oncolytic viruses in a manner similar to mir-199 . another mirna - based therapeutic strategy is the design of mirna panels that targets a particular protein to modulate inflammatory response and hcc development . the association between the ikk complex and rap1 is important for nf-b activation because rap1 serves as an adaptor to recruit ikks and aids in phosphorylation of the nf-b p65 subunit . 5-fluorouracil is a widely used chemotherapy drug for advanced hcc patients , but many patients with hcc are highly resistant to this chemotherapy regimen . a set of 4 rap1-mirnas was designed to knock down rap1 , leading to a significant increase in the sensitivity of hepg2 cells to 5-fluorouracil - induced apoptosis . a number of mirnas can be induced or inhibited during hepatitis because of viral or non - viral infections . some mirnas are also activated or repressed when inflammatory signaling pathways , such as nf-b and jak / stat3 signaling , can not be turned off . in turn , mirnas that might function as oncogenes or tumor suppressors can promote or suppress both inflammatory cascades in hepatocarcinogenesis . this function indicates that mirnas are novel regulators or mediators of liver inflammation and hepatocarcinogenesis ( figure 1 ) . the tumor microenvironment consists of many types of cells including tumor invading immune - cells , fibroblasts , vascular endothelial cells , lymphatic cells , and stromal components . given that numerous components of tumor microenvironment are responsible for the initiation , development , and progression of human cancer , the dysregulated mirnas from tumor specimens might originate from different types of cells in the tumor microenvironment and function in human carcinogenesis by multiple ways . the next investigations should focus on the origin , location , action , and multi - function of the dysregulated mirnas in initiation , development , and progression of hcc . additionally , any study of cell lines might run the risk of false discovery . the results from cell lines can not reflect the full aspects of the pathological process of hcc . the next investigations on the exact role of mirnas in inflammation - associated hcc should be verified in patients and reliable hcc models . a simplified sketch illustrates the development of liver inflammation and hcc and shows the key nodes in which aberrantly expressed mirnas participate in inflammation - associated hepatocarcinogenesis . the 5-year overall survival of hcc is lower than 10% worldwide because many patients are diagnosed at advanced stages . therefore , discovering novel viable biomarkers for hcc early diagnosis is urgent . in the last decades , numerous pieces of evidence highlighted the regulatory roles of mirnas in inflammation - associated hcc . evidence also indicated that the dysregulated mirnas might be useful as biomarkers for liver inflammation and hcc progression . in 2015 , mirnas were screened in a large - scale , multicenter , retrospective longitudinal study to explore effective and reliable strategies for monitoring at - risk populations that suffers from chronic inflammation as well as detect hcc at an early stage . serum mir-29a , mir-29c , mir-133a , mir-143 , mir-145 , mir-192 , and mir-505 might act as biomarkers in hcc because they are in significantly higher content in hcc compared with healthy controls or those with chronic inflammation . the classifier composed of these seven mirnas could be more sensitive than -fetoprotein in distinguishing patients with hcc from those who are cancer - free . the distinction requires large - scale , multicenter , independent investigations to confirm its feasibility . several studies illustrated the potential of mirnas as therapeutic targets to prevent or treat hcc . however , additional profiles and detailed mechanisms of mirna functions are required to provide a better understanding of their role in liver inflammation and hepatocarcinogenesis . the additional data will facilitate the application of more mirnas into prevention and therapy for inflammation - associated hcc . this work was supported by grants from the national key basic research program ( grant no . 2013cb910504 ) , and the national natural science foundation of china ( grant no . 81125016 and 81472565 ) .